METHODS, SYSTEMS, AND COMPOSITIONS RELATING TO MiRNA-146a

ABSTRACT

Some embodiments comprise methods, systems, and compositions to promote, improve and/or increase neuronal differentiation, oligodendrocyte differentiation, or neurological outcome or function in a patient in need thereof. Some embodiments also comprise the administration a composition comprising a pharmaceutically effective amount of one or more of a group comprising microRNA-146a, exosomes comprising microRNA-146a, a promoter of microRNA-146a expression, a microRNA-146a thymosin beta 4, and a phosphodiesterase 5 inhibitor to treat neurological conditions, diseases, or injuries in mammals, including in human beings.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 14/761,442, filed Jul. 16, 2015, which claims thebenefit of and priority to PCT Application No. PCT/US2014/012532, filedon Jan. 22, 2014, which claims the benefit of and priority to U.S.Provisional Patent Application Ser. No. 61/754,279, filed Jan. 18, 2013,all of the aforementioned disclosures are hereby incorporated byreference in their entireties.

SUPPORT

Some of the work described herein was supported by NIH grants R01AG038648, R01 NS075156, and R01 AG037506. The government may havecertain rights in some of the embodiments.

SEQUENCE LISTING

This application incorporates by reference in its entirety the SequenceListing entitled “25824-412469_ST25.txt” (4.25 KB), which was created onMay 16, 2017, and filed electronically herewith.

TECHNICAL FIELD

Without limitation, some embodiments comprise methods, systems, andcompositions relating to treatment of neurological conditions, disease,or injury, and the use of same in the research, diagnosis, and treatmentof such conditions, disease, or injury.

BACKGROUND

Brain disease, injury, or damage, as some non-limiting examples, strokeand traumatic brain injury, often induces long term neurologicaldeficits. Demyelination and remyelination are processes in which myelinsheaths are lost from around axons and later restored to demyelinatedaxons, respectively. These processes are involved in brain injury and inneurodegenerative diseases. Currently, there are no effective treatmentsavailable for improvement of neurological function after stroke, braininjury and neurodegenerative diseases.

Reduction of neurogenesis and loss of myelinating oligodendrocytesexacerbate neurological deficits. Diabetes affects an estimated 346million people world-wide and the vast majority of diabetics havenon-insulin-dependent type II diabetes. Peripheral neuropathy is one ofthe most common and disabling complications of diabetes mellitus. Thereis currently no effective treatment for preventing the development orreversing the progression of diabetic neuropathy.

SUMMARY

The following examples of some embodiments of the invention are providedwithout limiting the invention to only those embodiments describedherein and without waiving or disclaiming any embodiments or subjectmatter:

Some embodiments provide methods, systems, and compositions forpromoting, increasing, and/or improving neuronal differentiation,oligodendrocyte differentiation, and/or neurological outcome or functionin a patient in need thereof, including in mammals, and specifically inhuman beings. Some embodiments comprise the administration of acomposition comprising a pharmaceutically effective amount of one ormore of a group comprising, or consisting of, microRNA-146a, a promoterof microRNA-146a expression, exosomes containing or enriched withmicroRNA-146a, a microRNA-146a mimic, thymosin beta 4, and aphosphodiesterase 5 inhibitor to a patient in need of treatment for aneurological condition, disease, or injury.

Some embodiments provide a medicament comprising a pharmaceuticallyeffective amount of one or more of microRNA-146a, a promoter ofmicroRNA-146a expression, exosomes containing or enriched withmicroRNA-146a, a microRNA-146a mimic, thymosin beta 4, and aphosphodiesterase 5 inhibitor, and/or use of such a medicament intreating a patient with respect to the patient's neurological condition,disease, or injury, including but not limited to, in conjunction withstroke.

BRIEF DESCRIPTION OF DRAWINGS

Some embodiments will now be described, by way of example only andwithout waiver or disclaimer of other embodiments, with reference to theaccompanying drawings, in which:

FIG. 1 is comprised of images and data representations showing theeffect of miR-146a on proliferation and differentiation of neuralprogenitor cells.

FIG. 2 is comprised of images and data representations showing theeffect of miR-146a on differentiation of oligoprogenitor cells (“OPCs”).

FIG. 3 is comprised of images and data representations showing thatstroke upregulates miR-146a in SVZ neural stem cells.

FIG. 4 is comprised of data representations showing the effect ofmiR-146a treatment on neurological outcome after stroke.

FIG. 5 is comprised of images and data representations showing theeffects of expression of miR-146a and caspase 3 and neurologicalfunction in diabetic mice (db/db mice).

FIG. 6 is comprised of data representations showing the effect ofmiR-146a on cultured DRG neurons.

FIG. 7 is comprised of images showing immunostaining of primary ratembryonic OPCs.

FIG. 8 is comprised of data representations showing microRNA analysis ofmiR-146a in OPCs after Tβ4 treatment by qrtPCR.

FIG. 9 is comprised of images showing Western blot analysis ofdownstream signaling mediators of TLR in OPCs after Tβ4 treatment.

FIG. 10 is comprised of data representations showing quantitativeanalysis of expression of IL-1 receptor-associated kinase 1 (“IRAK1”),tumor necrosis receptor associated factor 6 (“TRAF6”), myelin basicprotein (“MBP”), phosphorylated ERK1 (“P-ERK1”), ERK1, phosphorylatedJNK1 (“P-JNK1”), JNK1, phosphorylated c-JUN (“P-c-JUN”), c-JUN,phosphorylated p38MAPK “(P-p38MAPK”) and p38MAPK at the protein levelafter Tβ4 treatment.

FIG. 11 is comprised of images and data representations showingapplication of LPS inhibitor polymyxin B for analysis of MBP expressionafter T34 treatment to test for confounding factor LPS contamination inTβ4.

FIG. 12 is comprised of images showing the effect of miR-146a andanti-miR-146a transfection on downstream signaling mediators of TLR.

FIG. 13 is comprised of a data representation showing QrtPCR analysis ofMBP and p38MAPK in OPCs.

FIG. 14 is comprised of images showing the effect of Tβ4 treatment andtransfection with miR-146a, anti-miR-146a and Tβ4siRNA on MBP expressionand downstream signaling mediators of TLR in primary rat embryonic OPCs.

FIG. 15 is comprised of images showing Western blot analysis of MBP anddownstream signaling mediators of TLR after Tβ4 treatment andtransfection with miR-146a, anti-miR-146a and Tβ4siRNA in the mouse OPCcell line-N20.1.

FIG. 16 is comprised of data representation showing quantitativeanalysis of expression of MBP, IRAK1, TRAF6, p38MAPK and phosphorylatedP-p38MAPK at the protein level.

FIG. 17 is comprised of images showing immunohistochemistry of MBP inprimary rat embryonic OPCs mouse N20.1 cells.

FIG. 18 is comprised of data representations showing quantitativeanalysis of MBP positive cells in primary rat embryonic OPCs and mouseN20.1 cells.

FIG. 19 depicts a timescale for performing the experiments related toadministration of miR-146a exosomes to treat diabetic neuropathy in malediabetic mice (db/db).

FIG. 20A depicts a bar graph illustrating results of real-time RT-PCRdata showing levels of serum miR-146a in the treated and untreatedanimals.

FIG. 20B depicts a bar graph illustrating results of real-time RT-PCRdata showing levels of sciatic nerve miR-146a in the treated anduntreated animals.

FIG. 21A depicts a line graph illustrating the effect of exosomescontaining miR-146a microRNA on the sciatic nerve conduction velocity intreated and untreated animals.

FIG. 21B depicts a line graph illustrating the effect of exosomescontaining miR-146a microRNA on the thermal hyperalgesia effects inducedin treated and untreated diabetic mice.

FIG. 21C depicts a line graph illustrating the effect of exosomescontaining miR-146a microRNA on the tactile allodynia effects indiabetic mice.

FIG. 22A depicts semi-thin toluidine blue-stained transverse sections ofsciatic nerves derived from non-diabetic mice (dm).

FIG. 22B depicts semi-thin toluidine blue-stained transverse sections ofsciatic nerves derived from diabetic mice (db) treated with saline.

FIG. 22C depicts semi-thin toluidine blue-stained transverse sections ofsciatic nerves derived from diabetic mice treated with miR-146a microRNAcontaining exosomes.

FIG. 22D depicts a table providing quantitative data ofhistomorphometric parameters of sciatic nerves from diabetic andnon-diabetic mice treated or untreated with miR-146a microRNA containingexosomes.

FIG. 23 depicts photomicrographs of intraepidermal nerve fibers (IENF)in skin of the foot-pads identified by the antibody against PGP9.5 ofdiabetic and non-diabetic mice treated or untreated with miR-146amicroRNA containing exosomes.

FIG. 24 depicts a bar graph depicting quantitative analysis of theeffects of miR-146a microRNA containing exosomes on the number of PGP9.5immunoreactive intraepidermal nerve fiber in diabetic mice (db+exomiR-146a) as shown in FIG. 23.

FIG. 25 depicts a bar graph representing levels of miR-146a microRNAafter intranasal administration of cerebral endothelial cell derivedexosomes (1×10⁹ particles/administration) in neural stem cells of Dicerknockout mice (n=10) compared to Dicer knockout mice treated with saline(n=12).

DETAILED DESCRIPTION

The following examples of some embodiments of the invention are providedwithout limiting the invention to only those embodiments describedherein and without waiving or disclaiming any embodiments or subjectmatter.

Some embodiments provide methods, systems, and compositions forpromoting, increasing, and/or improving neuronal differentiation,oligodendrocyte differentiation, and/or neurological outcome orfunction, and/or the treatment, prevention and/or amelioration of one ormore symptoms of a neurological disease, injury, disorder, or conditionin a patient in need thereof, including in mammals, and specifically inhuman beings. Some embodiments comprise the administration of acomposition comprising a pharmaceutically effective amount of one ormore of a group comprising, or consisting of, microRNA-146a, exosomescontaining or enriched in miR-146a, a promoter of microRNA-146aexpression, a microRNA-146a mimic, thymosin beta 4, and aphosphodiesterase 5 inhibitor to a patient in need of prevention and/ortreatment of a neurological condition, disease, or injury. As usedherein, the term “microRNA 146a” is also referred to herein as“miR-146a” and “miRNA-146a”, and are used interchangeably herein. Someembodiments provide a medicament comprising a pharmaceutically effectiveamount of one or more of microRNA-146a, exosomes containing or enrichedin microRNA-146a, a promoter of microRNA-146a expression, amicroRNA-146a mimic, thymosin beta 4, and a phosphodiesterase 5inhibitor, and/or use of such a medicament in treating a patient withrespect to the patient's neurological condition, disease, or injury,including but not limited to, in conjunction with stroke, traumaticbrain injury, neuropathy, for example, peripheral neuropathy, diabeticneuropathy, Multiple Sclerosis, Parkinson's Disease, Alzheimer's Diseaseand other neurological diseases, injuries, disorders and conditionsexemplified herein.

In accordance with some embodiments, without limitation, we havediscovered unexpectedly that overexpression of microRNA-146a (SEQ ID NO:1, UGAGAACUGAAUUCCAUGGGUU) can therapeutically enhance neurogenesis(generation of new neurons) and oligodendrogenesis (generation of matureoligodendrocytes) and improve neurological outcome or function andtherefore be useful in the treatment of neurological conditions,disease, and injury.

Some embodiments comprise the packaging of exosomes with one or morespecies of miRNA at high concentrations, for example, miR-146a, andthese miRNAs are delivered into tissue in therapeutic doses. PackagedmiRNAs can be those that are endogenous to the producing cells, but areforced to express at higher levels, or may be artificially designedmiRNAs, introduced to suit the therapeutic need. Also, a combination ofmiRNAs can be packaged within the same exosomes.

In various embodiments, the present invention provides a method ofpreventing or treating a subject or patient suffering from aneurological disease, condition, disorder, or injury. The treatmentcomprises treating the patient in need thereof, with exosomes containingmiR-146a microRNA, for example, exosomes enriched in miR-146a microRNA.In one embodiment, the method for preventing or treating a subject witha neurological disease, condition, disorder, or injury comprisesadministering a therapeutically effective amount of exosomes containingor enriched in microRNA-146a to the subject in need thereof.

In some related embodiments the neurological disease, condition,disorder, or injury comprises dementia; brain injury, for example,traumatic brain injury; neuropathy, for example, peripheral neuropathy;stroke or multiple sclerosis. In another embodiment, an exemplary methodcomprises the steps of: harvesting exosomes containing miR-146a microRNAfrom a cell population capable of producing exosomes containing miR-146amicroRNA or media containing the cell population; and administering tothe subject or patient in need thereof, the harvested exosomes in apharmaceutically effective amount to treat the subject with theneurological disease condition, disorder, or injury. In someembodiments, the method optionally includes a step of confirming thepresence of the miR-146a microRNA in the harvested exosomes.

In other embodiments, the present invention provides a method oftreating a subject or patient suffering from a neurological disease,condition, disorder, or injury. The treatment comprises treating thepatient in need thereof, with cells that are capable of producingexosomes containing miR-146a microRNA. The method comprises the stepsof: providing a cell population capable of producing exosomes containingmiR-146a; confirming the presence of the miR-146a microRNA in the cellpopulation, and administering to the subject in need thereof, the cellpopulation capable of producing exosomes containing miR-146a microRNA ina pharmaceutically effective amount to treat the subject with respect tothe neurological disease or injury.

In some of these embodiments, the cells that are capable of producingexosomes containing miR-146a microRNA can include mammalian, forexample, human: stem cells, mesenchymal stromal cells, umbilical cordcells, endothelial cells, for example, cerebral endothelial cells,Schwann cells, hematopoietic cells, reticulocytes, monocyte-deriveddendritic cells (MDDCs), monocytes, B lymphocytes, antigen-presentingcells, glial cells, astrocytes, neurons, oligodendrocytes, spindleneurons, microglia, and mastocytes. As used herein, stem cells, forexample, can include stem cells from a mammalian subject, for example ahuman subject. In various embodiments, mammalian stem cells can include,without limitation, a progenitor cell, an embryonic stem cell, apluripotent stem cell, an induced pluripotent stem cell, a hair folliclestem cell, a hematopoietic stem cell, a very small embryonic like stemcell, a mesenchymal stem cell, an endometrial regenerative cell (ERC),and a progenitor cell. In various embodiments, the cell populationincludes mesenchymal stem cells, mesenchymal stromal cells, andendothelial cells, for example, cerebral endothelial cells.

In various embodiments, methods of the present invention may utilize theabove referenced cell populations that are derived from the subject tobe treated, or they may be derived from a member of the same species,for example, the cells may be allogeneic.

In various embodiments, the exosomes may be administered to the patientin need thereof intravenously, nasally, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intratracheally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, injection, infusion, continuous infusion,localized perfusion bathing target cells directly, via a catheter, via alavage, or stereotactically into neural tissue. In each of theseadministration modes, the exosomes or cell populations capable ofproducing such exosomes containing or enriched in miR-146a areadministered to the subject in need thereof in accordance with standardmedically approved methods.

In each of these embodiments, the exosomes may be administered intherapeutically effective amounts. The therapeutically effective amountof exosomes containing the miR-146a microRNA may be dependent on severalfactors known to those skilled in the art. The amount must be effectiveto achieve improvement, including but not limited to, decreased damageor injury, or improvement or elimination of one or more symptoms andother indicators as are selected as appropriate measures by thoseskilled in the art.

In some embodiments, neurological diseases, disorders, conditions and/orinjuries that are beneficially treated, and/or prevented upon treatmentwith effective amounts of exosomes comprising miR146a, or cellpopulation bearing exosomes comprising miR146a include, but are notlimited to: stroke, brain injury, for example, traumatic brain injury(TBI), central pontine, dementia, multiple sclerosis (MS) (together withthe similar diseases called idiopathic inflammatory demyelinatingdiseases), tumefactive multiple sclerosis, Solitary sclerosis, cognitivedecline from aging, Alzheimer's disease, Parkinson's disease, epilepsy,migraine, neuropathy, for example, peripheral neuropathy, Vitamin B12deficiency, myelinolysis, Tabes Dorsalis, transverse myelitis, Devic'sneuromyelitis optica, fulminant or acute idiopathicinflammatory-demyelinating disease, Marburg variant of multiplesclerosis, Baló's concentric sclerosis, Schilder's disease, acutedisseminated encephalomyelitis; transverse myelitis, optic neuritis,progressive multifocal leukoencephalopathy, acute hemorrhagicleukoencephalitis, acute disseminated encephalomyelitis, anti-myelinoligodendrocyte glycoprotein autoimmune encephalomyelitis,Leukodystrophy, adrenoleukodystrophy, adrenomyeloneuropathy, chronicinflammatory demyelinating polyradiculoneuropathy (CIDP), Guillain-Barresyndrome, chronic inflammatory demyelinating polyneuropathy, or anti-MAGperipheral neuropathy.

In some embodiments, the present invention provides a method of treatinga subject suffering from dementia, neuropathy, brain injury, forexample, traumatic brain injury, stroke or multiple sclerosis withexosomes containing miR-146a microRNA. In these exemplary methods, thesteps may include: administering to the subject in need thereof,exosomes containing miR-146a microRNA or exosomes enriched with miR-146amicroRNA in a pharmaceutically effective amount to treat the subjectwith the dementia, brain injury, neuropathy, stroke or multiplesclerosis. Optionally, the method for treatment also includes the stepsof harvesting exosomes containing miR-146a microRNA from a cellpopulation capable of producing the exosomes or media containing thecell population, and confirming the presence of the miR-146a microRNA inthe harvested exosomes.

In each of these exemplary methods, the exosome producing cellpopulation may include stem cells, mesenchymal stromal cells, umbilicalcord cells, endothelial cells, Schwann cells, hematopoietic cells,reticulocytes, monocyte-derived dendritic cells (MDDCs), monocytes, Blymphocytes, antigen-presenting cells, glial cells, astrocytes, neurons,oligodendrocytes, spindle neurons, microglia; or mastocytes. As usedherein, stem cells may include, a progenitor cell; a pluripotent stemcell, for example, an embryonic stem cell; an induced pluripotent stemcell; a hair follicle stem cell; a hematopoietic stem cell; a very smallembryonic like stem cell; a mesenchymal stem cell; an endometrialregenerative cell (ERC); or a neural progenitor cell.

In these methods, the cells used to treat or ameliorate one or moresymptoms associated with dementia, neuropathy, stroke or multiplesclerosis with exosomes containing miR-146a microRNA, the exosomescontaining the miR-146a may be obtained from autologous cellpopulations. In various embodiments of the above referenced methods oftreatment of dementia, neuropathy, stroke or multiple sclerosis withexosomes containing miR-146a microRNA, the exosomes may be administeredintravenously, nasally, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intratracheally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, injection, infusion, continuous infusion,localized perfusion bathing target cells directly, via a catheter, via alavage, or directly into neural tissue.

As used herein, it is believed that the oxygen deprivation (ischemia)and inflammation of the brain lead to several neurological diseases andinjuries which may be chronic and/or acute. In some embodiments,neurological inflammation can progress to demyelination of neurons.Demyelination is the act of demyelinating, or the loss of the myelinsheath insulating the nerves. When myelin degrades, conduction ofsignals along the nerve can be impaired or lost, and the nerveeventually withers. This leads to certain neurodegenerative disorderslike multiple sclerosis and chronic inflammatory demyelinatingpolyneuropathy.

Central nervous system (CNS) demyelination is a cause and consequence ofa variety of neurological diseases and especially exemplified by MS andcognitive decline from aging, which follow a relapsing-remitting butthen progressive course and a more protracted but progressive course,respectively. In both instances, these maladies involve increasedoxidative stress (OS), which damages brain cells of oligodendrocytelineage that are responsible for brain myelination, and production ofmyelination inhibitory factors including specific miRNAs.

According to an embodiment of the invention, the methods describedherein are useful in inhibiting the development of and/or treatingmultiple sclerosis. Multiple sclerosis (MS), also known as “disseminatedsclerosis” or “encephalomyelitis disseminata”, is an inflammatorydisease in which the fatty myelin sheaths around the axons of the brainand spinal cord are damaged, leading to demyelination and scarring aswell as a broad spectrum of signs and symptoms. Disease onset usuallyoccurs in young adults, and it is more common in women. It has aprevalence that ranges between 2 and 150 per 100,000.

Demyelination may also play an important role in the pathophysiology oftraumatic brain injury. In experimental studies, brain injuries havebeen shown to be accompanied by a loss of myelin.

Neonatal brain disorders are also associated with demyelination andfailure of remyelination. White matter injuries in the newborn brain,such as hypoxic ischemic encephalopathy and periventricular leukomalaciacan result in cerebral palsy and cognitive disability. Failure ofremyelination in such conditions contributes to permanent demyelinatedlesions. Methods of the present invention are provided to treat,ameliorate and diminish any of the symptoms of ischemic tissue injury inthe brain and the demyelination associated with brain diseases,disorders described above and below.

The present invention provides a method of treating or preventing aneurological disease or injury. In some embodiments, the neurologicaldisease or injury is a disease or injury that results from direct traumato the brain, an acute or chronic autoimmune insult, or an acute orchronic inflammation, either systemically, or locally in the brain. Insome embodiments, the neurological disease or injury is treated uponadministration of a therapeutically effective amount of exosomescontaining miR-146a or cells from a cell population capable of producingexosomes containing miR-146a. In some of these embodiments, said patientis administered a therapeutically effective amount of exosomescontaining miR-146a microRNA or cells from a cell population capable ofproducing exosomes containing miR-146a microRNA of the inventionconjointly with an immune system suppressor, and/or an anti-inflammatoryagent, for example, a glucocorticoid.

Exosomes containing miR-146a or cells from a cell population capable ofproducing exosomes containing miR-146a of the invention are capable ofresolving, diminishing, ameliorating or improving a symptom associatedwith inflammation occurring in the brain of the subject, preferably ahuman subject. Glucocorticoids are also known for their role in treatinginflammation. However, the full anti-inflammatory potential ofglucocorticoids is often clinically constrained as a monotherapy due tothe rate and severity of treatment-limiting adverse events thataccompany high or prolonged dosing regimens. For example, theadministration of glucocorticoids can result in side effects that mimicCushing's disease. These side effects and others associated withglucocorticoid use include increased appetite and weight gain, depositsof fat in the chest, face, upper back, and stomach, water and saltretention leading to swelling and edema, high blood pressure, diabetes,slow healing of wounds, osteoporosis, cataracts, acne, muscle weakness,thinning of the skin, increased susceptibility to infection, stomachulcers, increased sweating, mood swings, psychological problems such asdepression, and adrenal suppression and crisis.

Advantageously, treatment of a subject in need thereof having aneurologic disease or injury with a combination of a glucocorticoid andexosomes containing miR-146a or cells from a cell population capable ofproducing exosomes containing miR-146a of the invention enhances theanti-inflammatory properties of both classes of agents while reducingthe effects associated with high doses of glucocorticoids alone.

In methods of the invention, wherein a glucocorticoid is administeredconjointly with exosomes containing miR-146a or cells from a cellpopulation capable of producing exosomes containing miR-146a of theinvention, the glucocorticoid may be chosen from any glucocorticoidknown in the art. Glucocorticoids suitable for said conjointadministration include, but are not limited to, alclometasone,amcinonide, beclometasone, betamethasone, budesonide, ciclesonide,clobetasol, clobetasone, clocortolone, cloprednol, cortisone,cortivazol, deflazacort, desonide, desoximetasone, desoxycortone,dexamethasone, diflorasone, diflucortolone, difluprednate, fluclorolone,fludroxycortide, flumetasone, flunisolide, fluocinolone acetonide,fluocinonide, fluocortin, fluocortolone, fluorometholone, fluperolone,fluprednidene, fluticasone, formocortal, halcinonide, halometasone,hydrocortisone/cortisol, hydrocortisone aceponate, hydrocortisonebuteprate, hydrocortisone butyrate, loteprednol, medrysone,meprednisone, methylprednisolone, methylprednisolone aceponate,mometasone furoate, paramethasone, prednicarbate,prednisone/prednisolone, prednylidene, rimexolone, tixocortol,triamcinolone, ulobetasol, mometasone, fluticasone propionate,beclomethasone dipropionate, fluocinolone, flunisolide hemihydrate,mometasone furoate monohydrate, desoxymethasone, diflorasone diacetate,hydrocortisone acetate, difluorocortolone, fluorocortisone,flumethasone, flunisolide, fluorocortolone, prednisolone, prednisone,cortisol, 6a-methylprednisolone, alclometasone dipropionate,fluclorolone acetonide, fluocinolone acetonide, betamethasone benzoate,fluocoritin butyl, betamethasone dipropionate, fluocortolonepreparations, betamethasone valerate, fluprednidene acetate,flurandrenolone, clobetasol propionate, clobetasol butyrate,hydrocortisone, hydrocortisone butyrate, methylprednisolone acetate,diflucortolone valerate, flumethasone pivalate, or triamcinoloneacetonide, or pharmaceutically acceptable salts thereof.

In certain embodiments, the patient to be treated by a method of theinvention may already be receiving an anti-inflammatory drug (other thana glucocorticoid). In one preferred embodiment, the patient is alreadytaking a glucocorticoid, such as one of the glucocorticoids describedabove, and will continue to take that drug conjointly with a compositioncomprising exosomes containing miR-146a or cells from a cell populationcapable of producing exosomes containing miR-146a of the invention.Alternatively, the exosomes containing miR-146a or cells from a cellpopulation capable of producing exosomes containing miR-146a of theinvention may be used as a replacement for the previously administeredanti-inflammatory, anti-autoimmune or immune suppressing drug, forexample, an anti-CD40L antibody, methotrexate, hydrocortisone,prednisone, prednisolone, methylprednisolone, betamethasone, VERIPRED™,ORAPRED™, triamcinolone acetonide, AVONEX™ (interferon beta-1a),BETASERON™ (interferon beta-1b), COPAXONE™ (glatiramer acetate),EXTAVIA™ (interferon beta-1b), GLATOPA™ (glatiramer acetate (Copaxone 20mg dose)), PLEGRIDY™ (peginterferon beta-1a), REBIF™ (interferonbeta-1a), ZINBRYTA™ (daclizumab) AUBAGIO™ (teriflunomide), GILENYA™(fingolimod), AMPYRA® (Dalfampridine), TECFIDERA™ (dimethyl fumarate),LEMTRADA™ (alemtuzumab), NOVANTRONE™ (mitoxantrone), or TYSABRI™(natalizumab), or alternatively, all of these anti-inflammatory,anti-autoimmune or immune suppressing drugs may be co-administeredindividually or in combination with a composition comprising exosomescontaining miR-146a microRNA or cells from a cell population capable ofproducing exosomes containing miR-146a microRNA of the invention, eitherconcurrently or sequentially to treat the neurologic disease or injuryas exemplified herein.

In one embodiment, the method of treating or preventing a neurologicdisease or injury according to this invention may comprise theadditional step of conjointly administering to the patient anotheranti-inflammatory agent, such as, for example, a non-steroidalanti-inflammatory drug (NSAID), a mast cell stabilizer, or a leukotrienemodifier.

In certain embodiments, the use of a composition comprising exosomescontaining miR-146a or cells from a cell population capable of producingexosomes containing miR-146a of the invention and a glucocorticoid orother anti0-inflammatory agent in the treatment of a neurologic diseaseor injury does not preclude the separate but conjoint administration ofanother anti-inflammatory agent for example, a corticosteroid, anon-steroidal anti-inflammatory drug (NSAID), a mast cell stabilizer, ora leukotriene modifier.

In certain embodiments, the present invention provides a kit comprising:a) one or more single dosage forms of a composition containing exosomescontaining miR-146a or cells from a cell population capable of producingexosomes containing miR-146a of the invention; and b) instructions forthe administration of the exosomes containing miR-146a or cells from acell population capable of producing exosomes containing miR-146a of theinvention.

The present invention provides a kit comprising: a) a pharmaceuticalformulation (e.g., one or more single dosage forms) comprising exosomescontaining miR-146a or cells from a cell population capable of producingexosomes containing miR-146a of the invention; and b) instructions forthe administration of the pharmaceutical formulation e.g., for treatingor preventing a disorder or condition as discussed above, e.g., aneurologic disease or injury.

In certain embodiments, the kit further comprises instructions for theadministration of the pharmaceutical formulation comprising exosomescontaining miR-146a or cells from a cell population capable of producingexosomes containing miR-146a of the invention conjointly with a anotherimmune system modulator, an anti-inflammatory agent, for example acorticosteroid, a non-steroidal anti-inflammatory drug (NSAID), a mastcell stabilizer, or a leukotriene modifier or combinations thereof asmentioned above. In certain embodiments, the kit further comprises asecond pharmaceutical formulation (e.g., as one or more single dosageforms) comprising an immune system modulator, an anti-inflammatoryagent, for example a corticosteroid, a non-steroidal anti-inflammatorydrug (NSAID), a mast cell stabilizer, a leukotriene modifier, or acombinations thereof as mentioned above.

EXAMPLES

The following examples of some embodiments are provided without limitingthe invention to only those embodiments described herein and withoutwaiving or disclaiming any embodiments or subject matter.

Example 1

In accordance with some embodiments, without limitation, we havediscovered unexpectedly that overexpression of microRNA-146a (also“miR-146a” or “miRNA-146a”) (SEQ ID NO: 1, UGAGAACUGAAUUCCAUGGGUU) cantherapeutically enhance neurogenesis (generation of new neurons) andoligodendrogenesis (generation of mature oligodendrocytes) and improveneurological outcome or function and therefore be useful in thetreatment of neurological conditions, disease, and injury.

Some embodiments comprise the use of miR-146a in: 1) augmentation ofnewly generated neurons and oligodendrocytes leading to the improvementof neurological function after stroke, and 2) reduction of dorsal rootganglion neuron death leading to improvements of sensory conduction indiabetic peripheral neuropathy.

In accordance with some embodiments, we evaluated whether miR-146aaffects adult neural stem cells to differentiate into neurons andoligodendrocytes. Primary neural stem cells isolated from thesubventricular zone (“SVZ”) of the lateral ventricle of the adult ratwere employed. Using a neurosphere assay, we examined the effect ofmiR-146a on neural stem cell proliferation and differentiation. Neuralstem cells were transfected with miR-146a mimics and cultured at adensity of 10 cells/μl in growth medium for 3 days. Bromodeoxyuridine(BrdU, 30 μg/ml, Sigma Aldrich), the thymidine analog that isincorporated into the DNA of dividing cells during S-phase, was added 18h prior to the termination of incubation. FIG. 1 shows the effect ofmiR-146a on proliferation and differentiation of neural progenitorcells. Panels A and B of FIG. 1 show BrdU immunoreactive cells aftertransfection with miRNA mimic controls and miR-146a mimics. Panels C-Dand E-F show Tuj1 and O4, respectively, immunoreactive cells aftertransfection with miRNA mimic controls and miR-146a mimics. Scale bar=20μm. N=3, p<0.05. We found that miR-146a mimics significantly (p<0.05)decreased the number of BrdU-positive cells (FIGS. 1A and B) compared tothe cells transfected with miRNA mimic controls, indicating thatexogenous miR-146a suppresses neural stem cell proliferation.

To evaluate the effect of miR-146a on neural stem cell differentiation,neural stem cells transfected with miR-146a mimics or mimic controlswere cultured under differentiation media containing 2% fetal bovineserum without growth factors. Every 4 days, half of the medium wasreplaced with fresh medium. Incubation was terminated 10 days afterplating. Immunostaining analysis revealed that introduction of miR-146astrikingly increased the percentage of Tuj1 (a marker of neuroblasts)positive cells (FIGS. 1C and D, n=3, p<0.05), but did not significantlyaffect the percentage of GFAP (a marker of astrocytes) positive cellscompared with the cells transfected with mimic controls. In addition,transfection of miR-146a mimics increased the number of Oligodendrocyte4 (O4) positive cells (FIGS. 1E and 1F), indicating that miR-146ainduces neural stem cells to differentiate into neurons andoligodendrocytes.

Our work demonstrated that miR-146a promotes neuronal andoligodendrocyte differentiation of adult neural stem cells.

In addition to neural stem cells, oligodendrocyte progenitor cells(“OPCs”) also differentiate into mature oligodendrocytes. We alsoexamined the effect of miR-146a on OPC differentiation. OPCs isolatedfrom embryonic day 18 rat embryos were used. We transfected OPCs withmiR-146a mimics. FIG. 2 shows the effect of miR-146a on differentiationof OPCs. Immunocytochemistry (FIG. 2A) shows O4 and MBP positive cellsin miRNA mimic control and miR-146a mimic groups. FIGS. 2B and C arequantitative data of O4 and MBP positive cells in different groups. FIG.2D is Western blot data showing levels of CNPase, MBP, PLP, PDGFRα andNG2 in OPCs transfected with miRNA mimic control, miR-146a mimics,inhibitor control, and miR-146a inhibitor. β-actin was used as aninternal control. *p<0.05, N=3/group. Scale bar=20 um.Immunocytochemistry analysis revealed that miR-146a mimics resulted in asignificant increase in the number of myelin basic protein (MBP, amarker of mature oligodendrocytes) positive cells (FIGS. 2A-C).Furthermore, Western blot analysis showed that miR-146a mimics robustlyincreased protein levels of MBP, proteolipid protein (“PLP”), and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (“CNPase”), all of them aremarkers of mature oligodendrocytes (FIG. 2D). In contrast, miR-146amimics considerably decreased oligodendrocyte progenitor cell proteinlevels, PDGFRa and NG2 (FIG. 2D). Our results indicate that exogenousmiR-146a promotes differentiation of oligodendrocyte progenitor cellsinto mature oligodendrocytes.

We also investigated the effect of endogenous miR-146a onoligodendrocyte maturation by transfecting OPCs with miRNA inhibitors.Attenuation of endogenous miR-146a significantly reduced the number ofMBP+ cells (FIG. 2C). Western blot analysis showed that inhibition ofmiR-146a decreased protein levels of MBP, PLP and CNPase, but increasedprotein levels of PDGFRa and NG2 (FIG. 2D). Our observations indicatethat endogenous miR-146a is required for oligodendrocyte maturation.

Collectively, our work demonstrated that miR-146a promotes OPCs todifferentiate into mature oligodendrocytes.

We also examined miR-146a levels post-stroke. FIG. 3 summarizes ourresults which show that stroke upregulates miR-146a in SVZ neural stemcells. FIGS. 3A and B show SVZ cells before (FIG. 3A) and after (FIG.3B) laser capture microdissection (“LCM”). Real-time RT-PCR analysis(FIG. 3C) shows miR-146a levels in non-ischemic (“non-MCAO”) andischemic (“MCAO”) neural stem cells isolated by LCM. LCM. N=6rats/group. Immunocytochemistry analysis (FIGS. 3D and E) shows Tuj1positive neuroblasts in non-ischemic (“non-MCAO”) and ischemic (“MCAO”)neural stem cells as well as ischemic neural stem cells transfected withmiR-146a inhibitor (MCAO+miR-146a inhibitor). N=3/group, *p<0.05.

We first isolated the neural stem cells from the SVZ of adultnon-ischemic rats and from rats subjected to middle cerebral arteryocclusion (“MCAO”) using laser capture microdissection (FIGS. 3A and B).Then, using real-time RT-PCR, we measured miR-146a levels in SVZ neuralstem cells and found that stroke substantially increased miR-146a levels(FIG. 3C). When SVZ neural stem cells were cultured in differentiationmedium, neural stem cells isolated from ischemic SVZ exhibitedconsiderable increases in neuroblasts measured by Tuj1 positive cellscompared to non-ischemic neural stem cells (FIGS. 3D and E).Transfection of miR-146a inhibitor into neural stem cells significantlysuppressed ischemia-increased neuroblasts (FIG. 3E), compared withnon-ischemic neural stem cells. Our data indicate that miR-146a mediatesstroke-induced neurogenesis.

Neurogenesis and oligodendrogenesis are related to neurologicalfunction. We also examined whether in vivo elevation of miR-146aimproves neurological outcome after stroke. Young adult Wistar rats weresubjected to MCAO. Using an Alzet micro-osmotic pump (35 μg, 1 ul/hr,Alzet, Cupertino, Calif., USA), we intraventricularly infused themiR-146a mimic oligonucleotides (e.g., available from Thermo Scientific,Waltham, Mass., USA, or its affiliates as part of the miRIDIAN™ productline) into the ischemic lateral ventricle for 7 days starting 1 dayafter MCAO (n−4). Ischemic rats (n=7) that received intraventricularinfusion of miRNA mimic control (cel-miR-67, 35 μg, Thermo Scientific)were used as a control group. The cel-miR-67 is not expressed in rodentbrain. A modified neurological severity score (“mNSS”) was performed 1,3, 7, and 14 days after MCAO by a person who was blinded to thetreatment. We found that all rats exhibited severe neurological deficitsprior to (1 d after MCAO) the infusion of miR-146a mimics (FIG. 4A).Compared to miRNA control treatment, treatment with miR-146a mimicssignificantly improved neurological outcome measured by the mNSSstarting 7d and persisting to 14 days after stroke (FIG. 4A, p<0.05).

To examine whether intraventricular infusion of miR-146a mimics elevatesbrain levels of miR-146a, we measured miR-146a and cel-miR-67 levels.FIG. 4 shows the effect of miR-146a treatment on neurological outcomeafter stroke. FIG. 4A shows that the administration of miR-146a mimicsat 24 hours after stroke improved neurological function measured by mNSS7 and 14 days after MCA occlusion compared with the control mimictreatment group. N=7 rats in control mimic group and N=4 in miR-146mimic group. FIG. 4B demonstrates that injection of cel-miR-67 mimicssubstantially increased cel-miR-67 expression in ipsilateral SVZ neuralprogenitor cells compared with that in contralateral SVZ cells. FIG. 4Cshows that the expression of miR-146a was significantly increased afterthe injection of mimic control—cel-miR-67 and further upregulated afterthe injection of miR-146a mimics in ipsilateral SVZ. Panels B and C areTaqman real-time RT-PCR data. N=3/group, *p<0.05, **p<0.01.

Briefly, total RNAs were extracted from SVZ tissues of ischemic ratstreated with miR-146a or cel-miR-67. Levels of these miRNAs weremeasured using real-time RT-PCR. We found that intraventricular infusionof cel-miR-67 significantly increased cel-miR-67 levels in theipsilateral SVZ tissue compared to that in the contralateral SVZ (FIG.4B), while miR-146a levels in the ipsilateral SVZ were 5.1 fold comparedto the contralateral SVZ (FIG. 4C). This elevated miR-146a level in theipsilateral SVZ after infusion of cel-miR-67 is comparable to the levelobserved in the ischemic SVZ of rats without the cel-miR-67 treatment(FIG. 2C). Thus, the increased miR-146a detected after infusion ofcel-miR-67 is likely induced by ischemia but not by cel-miR-67. However,intraventricular infusion of miR-146a mimics increased miR-146a levelsof the ipsilateral SVZ from 1.0 to 9.7 fold compared to the levels inthe contralateral SVZ (FIG. 4C). We did not detect cel-miR67 signals (Ctvalues above 40) in rats treated with miR-146a. These findings indicatethat the intraventricular infusion of miR-146a mimics specificallyelevate brain miR-146a levels.

Collectively, these data indicate that elevation of brain miR-146alevels by exogenous miR-146a mimics considerably improves neurologicaloutcomes after stroke.

Diabetes induces downregulation of miR-146a in dorsal root ganglion(“DRG”) neurons and peripheral neuropathy. Elevation of miR-146a byphosphodiesterase 5 inhibitors, as a nonlimiting example, sildenafil,improves neurological outcomes in diabetic peripheral neuropathy.

DRG neurons play an important role in sensory conduction. Diabeticperipheral neuropathy is characterized by the loss and/or degenerationof neurons and the slowing of nerve conduction velocities. Using a mousemodel of type II diabetes which develops severe peripheral neuropathy,we measured miR-146a expression in DRG neurons. In situ hybridizationand real-time RT-PCR analyses revealed that miR-146a signals weresubstantially reduced in DRG neurons of type II diabetic mice comparedthat in non-diabetic mice (FIG. 5). FIG. 5 shows expression of miR-146aand caspase 3 and neurological function in diabetic mice (db/db mice).In situ hybridization (FIG. 5A to C) shows miR146a signals (arrows) inrepresentative DRG neurons of non-diabetic mouse (FIG. 5A, dm), diabeticmouse (FIG. 5B, db/db) and diabetic mouse treated with sildenafil (FIG.5C, db/sil). FIG. 5D shows quantitative data of miR-146a levels in thesethree population mice measured by real-time RT-PCR. Immunofluorescentstaining (FIG. 5E to G) shows caspase 3 positive DRG neurons innon-diabetic (FIG. 5E, dm), diabetic (FIG. 5F, db) and diabetic micetreated with sildenafil (FIG. 5G, db/sil). FIG. 5H is quantitative dataof caspase 3 positive cells. FIGS. 5I and J are neurological functionmeasured by motor nerve conduction velocity (MCV, FIG. 5I) and sensorynerve conduction velocity (“SCV”, FIG. 5J). Red, blue, and black linesrepresent non-diabetic mice, diabetic mice treated with saline, anddiabetic mice treated with sildenafil, respectively. *P<0.01 versus thenondiabetic group. #P<0.05 versus the diabetic group treated withsaline. n=10/group. DRG neurons with reduction of miR-146a exhibitedapoptosis measured by cleaved caspase 3 immunoreactive cells (FIG. 5).Elevation of miR-146a in DRG neurons by sildenafil rescueddiabetes-induced DRG neuron death and improved neurological outcomes(FIG. 5).

In vitro, hyperglycemia significantly suppressed miR-146a expression incultured primary DRG neurons, which was associated with DRG neuron death(FIG. 6). FIG. 6 shows the effect of miR-146a on cultured DRG neurons.FIG. 6A is real-time RT-PCR data showing miR-146a levels in DRG neuronscultured under normal glucose (N) and high glucose (H) conditions.Substantial reduction of miR-146a was detected under high glucosecondition (n=6, *p<0.05). FIG. 6B shows percentage of TUNEL positive DRGneurons under normal glucose (N), high glucose (H), and high glucosewith miR-146a mimics (H/miR). * and # p<0.05 vs. normal and highglucose, respectively, n=6/group. FIG. 6C shows percentage of TUNELpositive DRG neurons under normal glucose (N) and normal glucose withsiRNA against miR-146a (N/siR). Attenuation of endogenous miR-146a withsiRNA against miR-146a significantly (p<0.05, n=6/group) increased TUNELpositive cells. Treatment of DRG neurons with miR-146a mimics completelyabolished hyperglycemia-induced reduction of miR-146a and neuronal death(FIG. 6). In addition, attenuation of endogenous miR-146a in DRG neuronsby siRNA against miR-146a resulted in considerable increases in neuronaldeath measured by TUNEL positive cells (FIG. 6).

Collectively, our in vivo and in vitro data indicate that downregulationof miR-146a in DRG neurons by diabetes induces neuronal death, whereaselevation of miR-146a levels suppresses diabetes-induced DRG neurondeath, leading to improvement of neurological outcomes. Our dataindicates that elevation of miR-146a levels in the ischemic brainsignificantly improves neurological outcomes after stroke and that,without limitation to any specific mechanism, enhancement ofneurogenesis and oligodendrogenesis by miR-146a is likely a mechanismunderlying the improved neurological outcome. Moreover, elevation ofmiR-146a in DRG neurons in diabetic peripheral neuropathy substantiallyreduces DRG neuron death and improves neurological function. Thus, inaccordance with some embodiments, miR-146a is a therapeutic target fortreatment of neurological conditions, disease, or injury, including butnot limited to, stroke, brain injury, neurodegenerative diseases, andperipheral neuropathy.

Some embodiments comprise miR-146a as a therapeutic target to enhanceneurogenesis and oligodendrogenesis in adult neural stem cells and OPCs,which facilitates repair processes in injured brain and inneurodegenerative diseases. In addition, this therapeutic target reducesdiabetes-induced DRG neuron death and improves neurological function indiabetic peripheral neuropathy.

In some embodiments, without limitation, miR-146a increases neuronaldifferentiation of neural progenitor cells, promotes maturation of OPCs,and improves neurological outcome post stroke. The results, which wereobtained in a widely accepted neural stem cell assay, were validated inan animal model of stroke. New neurons enhance neuronal functionincluding memory, while mature oligodendrocytes myelinate axons.Furthermore, miR-146a reduces diabetes-induced DRG neuron death andimproves neurological outcomes in diabetic peripheral neuropathy. Thus,in accordance with some embodiments, miR-146a can be used as a treatmentor target for therapeutic approaches against neurological conditions,disease, or injury, as nonlimiting examples, brain injuries, such asstroke and traumatic brain injury, neurodegenerative diseases, such asmultiple sclerosis and dementia and peripheral neuropathies, includingbut not limited to, diabetic peripheral neuropathy.

Example 2

In our work, we evaluated whether thymosin beta 4 (“Tβ4”) promotesdifferentiation of oligoprogenitor cells to oligodendrocytes in animalmodels of neurological injury. We discovered unexpectedly that Tβ4increased expression of microRNA-146a and suppressed expression ofproinflammatory cytokines of the Toll-like receptor (“TLR”) signalingpathway, and that Tβ4 suppresses the TLR proinflammatory pathway byupregulating miR146a, with implication for the promotion ofoligodendrogenesis for clinical purposes.

Tissue inflammation results from neurological injury and regulation ofthe inflammatory response is vital for neurological recovery. The innateimmune response system which includes the Toll-like receptor (“TLR”)proinflammatory signaling pathway regulates tissue injury. We evaluatedwhether that Tβ4 regulates the TLR proinflammatory signaling pathway.Since oligodendrogenesis plays an important role in neurologicalrecovery, we employed an in vitro primary rat embryonic cell model ofoligodendrocyte progenitor cells (“OPCs”) and a mouse N20.1 OPC cellline to measure the effects of Tβ4 on the TLR pathway. In brief summary,we grew cells in the presence of Tβ4 ranging from 25 to 100 ng/ml of(RegeneRx Biopharmaceuticals Inc., Rockville, Md.) for 4 days.

Quantitative real-time (“Qrt:) PCR and Western blot data demonstratedthat Tβ4 treatment increased expression of microRNA-146a (also“miR-146a”), a negative regulator of the TLR signaling pathway, in thesetwo cell models. Western blot analysis showed that Tβ4 treatmentsuppressed expression of IL-1 receptor associated kinase 1 (“IRAK1”) andTNF receptor-associated factor 6 (“TRAF6”), two proinflammatorycytokines of the TLR signaling pathway. Transfection of miR-146a intoboth primary rat embryonic OPCs and mouse N20.1 OPCs treated with Tβ4demonstrated an amplification of myelin basic protein (“MBP”) expressionand differentiation of OPCs into mature MBP expressing oligodendrocytes.Transfection of anti-miLR-146a nucleotides reversed the inhibitoryeffect of Tβ4 on IRAK1 and TRAF6 and decreased expression of MBP. Ourdata indicate that Tβ4 promotes OPC differentiation at least in part bysuppressing the TLR proinflammatory pathway via upregulating miR146a.

Tβ4 is a 5K Dalton, 43-amino acid peptide originally isolated from thethymus gland (SEQ ID NO: 2,Ac-Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu-Ile-Glu-Lys-Phe-Asp-Lys-Ser-Lys-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Glu-Lys-Asn-Pro-Leu-Pro-Ser-Lys-Glu-Thr-Ile-Glu-Gln-Glu-Lys-Gln-Ala-Gly-Glu-Ser).Improvement in neurological outcome is associated witholigodendrogenesis, i.e., differentiation of oligoprogenitor cells(“OPC”) into mature myelin secreting oligodendrocytes (“OL”).Oligodendrogenesis contributes to remyelination after neurologicalinjury by differentiation of OPCs into mature myelin expressing OL.

The innate immune system has been implicated in mediating theinflammatory response to cardiac injury and disease. Toll-like receptors(“TLRs”) are pattern recognition receptors that recognize conservedmolecular patterns of pathogens. In addition to pathogens, TLRs alsorecognize damage-associated molecular patterns (“DAMPs”) which aremolecular patterns of endogenous host debris released during cellularinjury or death. This debris can be extracellular matrix protein,oxidized proteins, RNA or DNA. Once recognition occurs, the TLRs arestimulated resulting in activation of many signaling pathways, includingthose pathways involving the mitogen activated protein kinases (“MAPKs”)and the nuclear factor NF-κB transcription factors. The MAPKs activateOL differentiation, and therefore TLR signaling may be involved inoligodendrogenesis as well as in regulating the inflammatory response.In addition, the TLR pathways are affected by miR-146, whichdownregulates proinflammatory cytokine production and activation ofinflammatory pathways. TLR4 is a well-studied TLR which mediates itsproinflammatory response through three proteins, IRAK1 (IL-1receptor-associated kinase 1), IRAK4 and TRAF6 (tumor necrosis receptorassociated factor 6). By targeting IRAK1 and TRAF6, miR-146 inhibitsNF-κB activation. We evaluated whether Tβ4 inhibits the TLRproinflammatory signaling pathway by specifically increasing miR-146a topromote differentiation of OPCs to MBP expressing OLs.

Experimental Procedures

All animal experiments were performed according to protocols approved bythe Henry Ford Hospital Institutional Animal Care and Use Committee.

Isolation of Primary Rat Embryonic OPCs

Primary rat embryonic OPCs were isolated and prepared according to themethod known to the skilled artisan. Briefly, on embryonic day 17, ratembryos were removed from a pregnant Wistar rat in a laminar flow hood.The cortices were dissected out by using microdissecting scissors,rinsed twice in Hank's buffered salt solution and dissociated afterdigesting with 0.01% trypsin and DNase at 37° C. for 15 min. Thedigested cells were washed twice, filtered through a 70 mm nylon cellstrainer and plated with DMEM containing 20% fetal bovine serum (FBS) inpoly-D-lysine coated T75 cell culture flasks (approximately 10 millioncells per flask). The cells grew to confluence for 10 days and then wereplaced on the shaker at 200 r.p.m. at 37° C. for 1 h to removemicroglial cells. Subsequently, the cells were left on the shaker for anadditional 18-20 h to collect OPCs. The collected OPCs were plated inuntreated Petri dishes for 1 h to remove contaminated microglia andastrocytes which attach to the Petri dish more efficiently than OPCs.The unattached OPCs were transferred onto poly-D,L-ornithine-coatedPetri dishes at a cell density of 10⁴ per/cm2 with a basal chemicallydefined medium (“BDM”) containing 10 ng/ml platelet-derived growthfactor-alpha (“PDGF-AA”) and 10 ng/ml basic fibroblast growth factor(“bFGF”) for 7-10 days.

Cell Culture, Transfection and Treatment with Tβ4

The mouse primary cultures of OPCs were conditionally immortalized bytransformation with a temperature-sensitive large T-antigen into a mouseOPC cell line-N20.1 (21). N20.1 cells were provided by Dr. AnthonyCampagnoni (University of California at Los Angeles). N20.1 cells weregrown and maintained in Dulbecco's modified Eagle's medium (“DMEM”)/F12with 1% fetal bovine serum (“FBS”) and G418 (100 μg/ml) at 37° C. ForN20.1 cells, transient transfections were performed with Nucleofectorkit according to the manufacturer's protocol (Amaxa, Germany). The cells(10⁶) were mixed with 1 μg plasmid DNA or 100 pmol of siRNA/oligonucleotides and pulsed according to the manufacturer's instruction. Thetransfected cells were immediately plated into Petri dishes with DMEMcontaining 1% FBS and incubated at 37° C. for days. Primary ratembryonic OPCs were transiently transfected with Lipofectamine(Invitrogen) overnight, according to the manufacturer's protocol (seeChew et al., J. Neuroscience 30(33): 11011-11027 (2010)). Amounts of DNAand siRNA/oligo nucleotides were used as recommended by themanufacturer. The control plasmid (pcDNA3) was used as a mocktransfected control for miR-146a expression vector transfection, andcontrol siRNA (Ambion, a random mixture of oligonucleotides) was used asa mock transfected control for both transfections with Tβ4siRNA andanti-miR-146a inhibitor nucleotides. The cells (10⁴ cells/cm²) weretreated with 0, 25, 50, 100 ng/ml of Tβ4 (RegeneRx BiopharmaceuticalsInc., Rockville, Md.). The cells were incubated at 37° C. and fed every2 days with fresh medium (DMEM containing 1% FBS for N20.1 cells and BDMfor primary rat embryonic OPCs) with and without Tβ4 for 4 days. To testfor LPS contamination in Tβ4, the cells were cultured in the presence ofthe LPS inhibitor polymyxin B (50 μg/ml) followed by treatment with Tβ4.Tβ4 (100 ng/ml) was boiled for 10 minutes in order to denature Tβ4protein and was used as a negative control. Transfected cells (2×10⁴cells/cm2) including mock transfected controls were treated with andwithout 100 ng/ml of Tβ4 (RegeneRx Biopharmaceuticals Inc., Rockville,Md.) for 4 days and fresh medium was provided at day 2 with/without Tβ4.

Quantitative Real Time PCR (“qrtPCR”)

The extraction of total RNA and preparation of cDNA were performed asknown to the skilled artisan. The QrtPCR amplification was done for 40cycles in the following thermal cycle: 95° C. for 30 s, 60° C. for 30 s,and 72° C. for 45 s using SYBR green (Life Technologies, Grand Island,N.Y.). The primer sequences were:

CNPase: Forward-  (SEQ ID NO: 3) 5T-TACTTCGGCTGGTTCCTGAC-3T 5T- Reverse- (SEQ ID NO: 4) T-GCCTTCCCGTAGTCACAAAA-3T 5T-  (m,r) MBP:Forward-  (SEQ ID NO: 5) ATGGCATCACAGAAGAGACCCTCA-3′ 5T-  Reverse- (SEQ ID NO: 6) TAAAGAAGCGCCCGATGGAGTCAA-3' 5'-  (m,r) p38 (r): Forward-(SEQ ID NO: 7) ATGACGAAATGACCGGCTAC-3' 5T-  Reverse- (SEQ ID NO: 8)ACAACGTTCTTCCGGTCAAC-31 5T-  p38 (m): Forward- (SEQ ID NO: 9)GCTGAACAAAGGGAGAGACG-3T 5T-  Reverse-  (SEQ ID NO: 10)TGCTTTCTCCCCAAATTGAC-3T 5T-  JNK1 (r): Forward-  (SEQ ID NO: 11)TTCAATGTCCACAGATCCGA-3T 5'-  Reverse-  (SEQ ID NO: 12)CTAACCAATTCCCCATCCCT-3T 5T-  JNK1 (m): Forward-  (SEQ ID NO: 13)GCCATTCTGGTAGAGGAAGTTTCTC-3' 5T-  Reverse-  (SEQ ID NO: 14)CGCCAGTCCAAAATCAAGAATC-3' 5T-  Jun (r): Forward-  (SEQ ID NO: 15)TGAAGCAGAGCATGACCTTG-3' 5T-  Reverse-  (SEQ ID NO: 16)CACAAGAACTGAGTGGGGGT-3' 5T-  Jun (m): Forward-  (SEQ ID NO: 17)CGCAACCAGTCAAGTTCTCA-3T Reverse-  (SEQ ID NO: 18)GAAAAGTAGCCCCCAACCTC-3T.

After qrtPCR, agarose gel electrophoresis was performed to verify thequality of the qrtPCR products. No secondary products were detected.Each sample was tested in triplicate and all values were normalized toGAPDH. Values obtained from three independent experiments were analyzedrelative to gene expression data using the 2^(−ΔΔCT) method.

Quantification of Mature miRNAs by Real-Time qrtPCR

The cDNA for each miR and TaqMan assay were performed in triplicateaccording to the manufacturer's protocol specified in Applied BiosystemsViiA™ 7 Real-Time PCR System (Applied Biosystem). Briefly, total RNA wasisolated with TRIzol (Qiagen). Reverse transcription reaction mixturecontained 1-10 ng total RNA, 5 U MultiScribe Reverse Transcriptase, 0.5mM each dNTPs, 1× Reverse Transcription buffer, 4 U RNase Inhibitor, andnuclease free water. The microRNA cDNA was performed by individualreverse transcription in the following thermal cycle 16° C. for 30 min,42° C. for 30 min, 85° C. for 5 mM, and then hold at 4° C. for TaqManassays. TaqMan assay was performed in 20 μl TaqMan real-time PCRreactions containing 1×TaqMan Universal PCR Master Mix No AmpErase UNG,1×TaqMan miRNA assay, 1.330 of undiluted cDNA, and nuclease free water.All values were normalized to U6 snRNA TaqMan miRNA control assay(Applied Biosystem) as the endogenous control. Values obtained fromthree independent experiments were analyzed relative to gene expressiondata using the 2^(−ΔΔCT) method.

Western Blot Analysis

Total protein extracts from the cells were prepared, as known to theskilled artisan. The protein extracts were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis for Western blot analysis.For Western blot analysis, rabbit antiserum for MBP (1:200; Dako,Carpinteria, Calif.), monoclonal antibodies (1:1000) for p38MAPK,phosphorylated p38MAPK, c-JUN, phosphorylated c-JUN (1:1000; Upstate,Charlottesville, Va., USA), rabbit polyclonal antibodies (1:1000) forJNK1 and phosphorylated JNK1 (Promega Corporation), goat polyclonalTLR2, rabbit polyclonal TLR4, mouse monoclonal IRAK1 antibodies(1:1000), rabbit polyclonal TRAF6 antibodies (1:1000) mouse monoclonalβ-actin antibodies (1:5000; Santa Cruz Biotechnology) and mousemonoclonal α-tubulin antibodies (1:5000; Sigma) were used. Donkeyanti-goat, anti-rabbit, and anti-mouse horseradish peroxidase (1:5000;Jackson ImmunoResearch Labs, West Grove, Pa., USA) were used assecondary antibodies. Each experiment was repeated at least three times.The protein bands were quantified based on histogram analysis relativeto gel loading marker α-tubulin.

Immunochemistry

Immunofluorescence staining was performed in N20.1 and primary ratembryonic OPC cells. These cells were fixed with 4% paraformaldehyde for1 h, washed with PBS, blocked with 1% serum for 1 h and incubated withmonoclonal antibodies of OPC marker—O4, (1:1000, Chemicon, Billerica,Mass., USA) and a polyclonal antibody against mature OL marker—MBP(1:200; Dako, Carpinteria, Calif.) at room temperature for one hour,rinsed with PBS and secondary antibodies labeled with cyaninefluorophore (Cy3—red fluorescence) for 1 h. The slides werecounterstained with 4′,6-diamidino-2-phenylindole (DAPI—bluefluorescence) and examined under Fluorescent Illumination Microscope(Olympus IX71/IX51, Tokyo, Japan). DAPI positive cells were consideredas total number of cells.

Statistical Analysis

Data were summarized using mean and standard deviation. To compare thedifferences between cell cultures with and without Tβ4 treatment, a onesample t-test or a two-sample t-test was used. For the comparisons ofQrtPCR of mRNA/GAPDH and Quantitative Real Time PCR (“qrtPCR”) ofmiR-146a/U6, controls were normalized to 1, so that one-sample t-testwas used for analysis. To compare the percentage of positive stainedcells out of the total number of cells between Tβ4 treatment andcontrol, a two-sample t-test was used. P-value<0.05 was consideredsignificant

Results

We discovered unexpectedly that Tβ4 increases expression of miR-146a inOPCs. We investigated the effect of Tβ4 treatment on the expression ofmiR-146a and miR-146a in primary rat embryonic OPCs (n=5) and in a mouseOPC cell line—N20.1 (n=5) by qrtPCR. Purity of rat primary OPCs used inthe experiments was confirmed by immunostaining for O4 and wasquantified by cell counting. The cell counting data showed that >90% ofthese cells were O4 positive. FIG. 7 shows immunostaining of primary ratembryonic OPCs. Primary rat embryonic OPCs were immunostained for O4labeled with fluorescence Cy3 and counter stained for nuclei with DAPI.The cells were quantified by counting as percentage of O4 positive cellswhen DAPI positive cells were considered as total number of cells (shownat the bottom). We found that Tβ4 treatment induced the expression ofmiR-146a in rat primary embryonic OPCs and mouse N20.1 cells in adose-dependent manner (FIG. 8). In contrast, Tβ4 treatment had no effecton miR-146a expression in rat primary embryonic OPCs and mouse N20.1cells (data not shown). FIG. 8 shows microRNA analysis of miR-146a inOPCs after Tβ4 treatment by qrtPCR. The total RNA samples were extractedfrom primary rat embryonic OPCs (left panel) and mouse OPC cellline—N20.1 (right panel) after the treatment with Tβ4 at the doseranging from 0 to 100 ng/ml (shown at the bottom) for microRNA analysisof miR-146a by qrtPCR. Note that expression miR-146a was increased in adose dependent manner in both OPCs. P<0.05 was considered assignificant. We also discovered unexpectedly that Tβ4 down regulates theintracellular TLR signaling pathway in OPCs. MiR-146a targets twoproinflammatory cytokines, IRAK1 and TRAF6, in the intracellular TLRsignaling pathway. We evaluated the effect of Tβ4 treatment on the TLRsignaling pathway in rat primary embryonic OPCs and mouse N20.1 cells.These cell cultures which demonstrated induction of miR-146a expressionafter Tβ4 treatment (FIG. 8) were utilized to analyze the expressionlevels of IRAK1, TRAF6 and MBP, the mature OL marker, by Western blot.Tβ4 treatment markedly reduced the expression levels of IRAK1 and TRAF6,and increased the expression level of MBP in a dose-dependent manner inrat primary embryonic OPCs (n=3) and mouse N20.1 OPCs (n=3) (FIG. 9).

FIG. 9 shows Western blot analysis of downstream signaling mediators ofTLR in OPCs after Tβ4 treatment. The protein samples were separated,transferred and analyzed from the primary rat embryonic OPCs (leftpanel) and mouse OPC cell line-N20.1 (right panel) after the treatmentwith Tβ4 at the dose ranging from 0 to 100 ng/ml (shown at the top) andanalyzed for different protein expressions. Migrations of proteins areshown at right. The loading of the samples were normalized with β-actinand α-tubulin. These data indicate that the TLR signaling pathway may beinvolved in Tβ4 mediated OL differentiation in primary rat embryonicOPCs and mouse N20.1 cells.

FIG. 10 shows quantitative analysis of expression of IRAK1, TRAF6, MBP,phosphorylated ERK1 (P-ERK1), ERK1, phosphorylated JNK1 (P-JNK1), JNK1,phosphorylated c-JUN (P-c-JUN), c-JUN, phosphorylated p38MAPK(P-p38MAPK) and p38MAPK at the protein level after Tβ4 treatment.Western blot data from the primary rat embryonic OPCs (O) and mouse OPCcell line—N20.1 (N) after treatment with Tβ4 at the dose ranging 0, 25,50 and 100 ng/ml were quantified based on histogram analysis in comparedto α-tubulin. The bar graph indicates relative protein expression incompared to α-tubulin. P<0.05 was considered as significant.

We discovered unexpectedly that downstream signaling of the MAPKs in Tβ4mediates oligodendrocyte differentiation. We evaluated the effect of Tβ4on MAPKs which are downstream of the TLR pathway. Expression of TLR2 andTLR4 were confirmed by Western blot analysis (FIG. 9). However,treatment with Tβ4 had no effect on expression of TLR2 and TLR4 (FIG. 9,FIG. 10). Western blot was performed to measure expression andphosphorylation of p38MAPK, ERK1, JNK1, and c-Jun after Tβ4 treatment(FIG. 9, FIG. 10). Tβ4 treatment induced expression and phosphorylationof p38MAPK, a known regulator of oligodendrocyte differentiation, in adose-dependent manner. In contrast, Tβ4 dose-dependently inhibited thephosphorylation of ERK1/2, JNK1 and c-Jun in primary rat embryonic OPCsand mouse N20.1 cells (FIG. 9, FIG. 10).

We also discovered that the effect of Tβ4 on the oligodendrocytedifferentiation marker MBP is independent on LPS contamination in Tβ4.To avoid confounding data because of possible LPS contamination in Tβ4,OPC and N20.1 cells were cultured in the presence of polymyxin B (50μg/ml) followed by Tβ4 treatment at the dose of 50 and 100 ng/ml for 4days. The qrtPCR data indicate that Tβ4 treatment induced the expressionof MBP in a dose-dependent manner even in the presence of polymyxin B(50 μg/ml) in rat OPC and N20.1 cells in both mRNA and protein levels(FIGS. 11A, B & C). In contrast, the boiled denatured Tβ4 (100 ng/ml)treatment had no effect on MBP expression (FIGS. 11A, B & C). FIG. 11shows the results of application of LPS inhibitor polymyxin B foranalysis of MBP expression after Tβ4 treatment to test for confoundingfactor LPS contamination in Tβ4. The total RNA and protein samples wereprepared from primary rat (n=3) embryonic OPCs and mouse OPC cellline—N20.1 which were cultured in presence of polymyxin B (50 μg/ml)followed by the treatment with Tβ4 at the dose of 50 and 100 ng/ml inthree independent experiments. Bar graph (A) indicates relative mRNAexpression in compared to control for MBP in primary rat embryonic OPCsand mouse N20.1 cells. The protein samples were analyzed by Western Blot(B). Loading of samples shown at the top were normalized with α-tubulin.Migrations of proteins were shown at right. The protein bands in Westernblot were quantified based on histogram analysis in compared toα-tubulin in Bar graph (C). P<0.05 was considered as significant. Thesedata indicate that induction of MBP was solely dependent on natural Tβ4and independent of LPS contamination.

We also determined the effect of miR-146a and anti-miR-146a ondownstream signaling mediators of TLR and MAPKs. We measured proteinexpression of IRAK1, TRAF6 and MAPKs in miR-146a overexpressing andmiR-146a knockdown primary rat embryonic OPCs (n=3) and mouse N20.1cells (n=3) (FIG. 12). Overexpression and knockdown of miR-146a weredetermined by quantitative analysis of miR-146a. After miR-146atransfection, miR-146a increased 51±5.3 fold in N20.1 cells andincreased 33.5±4.1 fold in rat OPCs. A decrease of 73.1±8.3 fold inN20.1 cells and 46.7±5.2 fold in rat OPCs was observed for miR-146aknockdown. Western blot analysis revealed that the miR-146a transfectioninhibited expression of IRAK1 and TRAF6 and increased expression andactivation of p38MAPK. In contrast, transfection with anti-miR-146ainhibitor nucleotides significantly inhibited the expression of MBP andphosphorylation of p38MAPK (FIG. 12). Expression of IRAK1, TRAF6,p-ERK1, p-JNK and p-c-Jun remained unchanged or slightly elevated. Thesedata indicate that miR-146a may be directly involved in OLdifferentiation by activation of the p38MAPK signaling pathway in ratprimary embryonic OPCs and mouse N20.1 cells. FIG. 12 is comprised ofimages showing the effect of miR-146a and anti-miR-146a transfection ondownstream signaling mediators of TLR. The primary rat embryonic OPCs(left panel) and mouse OPC cell line—N20.1 (right panel) weretransfected with control pcDNA3 vector, miR-146a expression (pcDNA3)vector, control siRNA (Ambion) containing a random mixture ofoligonucleotides for nucleotide control as a control for anti-miR-146anucleotides (shown at the top) and were lysed for protein extraction andWestern blot analysis. The loading of the samples were normalized withα-tubulin. Migrations of proteins are shown at right.

We also found that Tβ4 regulates miR-146a expression. To investigate themechanistic link between Tβ4 and miR-146a on MBP expression, we furtherinvestigated the effect of both Tβ4 and miR-146a on the TLR signalingpathways using primary rat embryonic OPCs (n=3) and the mouse OPC cellline N20.1 (n=3).

FIG. 13 shows the results of QrtPCR analysis of MBP and p38MAPK in OPCs.QrtPCR analysis of MBP and p38MAPK was performed in total RNA samplesextracted from the following transfected primary rat embryonic OPCs (RatOPCs) and mouse OPC cell line—N20.1 (shown at bottom). These cells weretransfected with control plasmid (plasmid control) and miR-146a vector(miR-146a transfection) followed by treatment without and with Tβ4 (100ng/ml) (miR-146a+Tβ4). These OPCs were also transfected withanti-miR-146a (anti-miR-146a) and Tβ4siRNA. P<0.05 was considered assignificant. FIG. 13 demonstrated a two-fold increase in mRNA MBPexpression in the miR-146a transfection and Tβ4 group in rat primaryembryonic OPCs and mouse N20.1 cells. However, a 10-fold increase inmRNA MBP expression was observed when miRR-146a transfected cells weregrown in the presence of Tβ4, suggesting that Tβ4 amplifies miR-146ainduced MBP expression. A similar but less robust result was observedwhen measuring p38MAPK.

FIG. 14 shows the effect of Tβ4 treatment and transfection withmiR-146a, anti-miR-146a and Tβ4siRNA on MBP expression and downstreamsignaling mediators of TLR in the primary rat embryonic OPCs: In theleft panel, the primary rat embryonic OPCs were transfected with controlpcDNA3 vector (Control vector), miR-146a expression vector (miR-146avector), control pcDNA3 vector followed by Tβ4 treatment (Tβ4 100 ng/ml)and miR-146a expression vector followed by Tβ4 (100 ng/ml) treatment(miR-146a+Tβ4) (shown at the top). In the right panel, the primary ratembryonic OPCs were transfected with control siRNA, anti-miR-146a,Tβ4siRNA, Tβ4siRNA+miR-146a and anti-miR-146a followed by Tβ4 (100ng/ml) treatment (anti-miR-146a+Tβ4) (shown at the top). These cellswere lysed for protein extraction and Western blot analysis. The loadingof the samples were normalized with α-tubulin. Migrations of proteinsare shown at right. MiR-146a transfection combined with Tβ4 treatmentmarkedly induced MBP expression in the OPCs. Tβ4 treatment failed toinduce MBP expression in the absence of miR-146a as well as miR-146atransfection has no effect on MBP expression in Tβ4 negative OPCs.

FIG. 15 shows the results of Western blot analysis of MBP and downstreamsignaling mediators of TLR after Tβ4 treatment and transfection withmiR-146a, anti-miR-146a and Tβ4siRNA in the mouse OPC cell line—N20.1:The left panel indicates N20.1 cells transfected with control pcDNA3vector (Control vector), miR-146a expression vector (miR-146a vector),control pcDNA3 vector followed by Tβ4 treatment (Tβ4 100 ng/ml) andmiR-146a expression vector followed by Tβ4 (100 ng/ml) treatment(miR-146a+Tβ4) (shown at the top). The right panel indicates N20.1 cellstransfected with control siRNA, anti-miR-146a, Tβ4siRNA,Tβ4siRNA+miR-146a and anti-miR-146a followed by Tβ4 (100 ng/ml)treatment (anti-miR-146a+Tβ4) (shown at the top). The loading of thesamples were normalized with α-tubulin. Migrations of proteins are shownat right. Note that marked induction of MBP was observed after miR-146atransfection combined with Tβ4 treatment in N20.1. Notice that eitherTβ4 treatment or miR-146a transfection had no effect on MBP expressionin the absence of miR-146a or Tβ4 in N20.1 cells.

Western blot demonstrated similar results at the protein level as shownin FIG. 14 (primary rat OPCs) and FIG. 15 (mouse N20.1 cells).Furthermore, knockdown of miR-146a or silencing of Tβ4 using Tβ4siRNA(transfection efficiency of Tβ4siRNA was 58.3±6.2 fold in rat OPCs and75.1±7.9 fold in N20.1 cells) inhibited MBP expression with no effect onthe proinflammatory expression of IRAK1 and TRAF6 or the MAPKs, p-ERK1,p-JNK1 and p-c-Jun when compared with control (FIGS. 14 and 15).Silencing Tβ4 using Tβ4siRNA in miR-146a overexpressing cells showedinhibition of IRAK1 and TRAF6 without an increase of MBP expressionsuggesting that Tβ4 may be necessary for MBP expression. In contrast,knockdown of miR-146a cells treated with Tβ4 showed no change in theexpression of MBP, IRAK1, TRAF6, p38 MAPK p-ERK1, p-JNK1, and p-c-Jun.These data indicate that miR-146a is a necessary component for Tβ4mediated MBP expression. A histogram summarizing the protein expressionsof MBP, IRAK1, TRAF6 and p38 MAPK in miR-146 overexpressed cells treatedwith Tβ4 is shown in FIG. 16. FIG. 16 shows the results of quantitativeanalysis of expression of MBP, IRAK1, TRAF6, p38MAPK and phosphorylatedp38MAPK (P-p38MAPK) at the protein level. Western blot data from theprimary rat embryonic OPCs (OPC) and mouse OPC cell line—N20.1 (N20.1)transfected with control vector and miR-146a expression vector followedby treatment with/without Tβ4 (100 ng/ml) were quantified based onhistogram analysis in compared to α-tubulin. Bar graph indicatesrelative protein expression in compared to α-tubulin (at left) for MBP,IRAK1, TRAF6, p38MAPK and phosphorylated p38MAPK (P-p38MAPK) (at bottom)in primary rat embryonic OPCs and mouse N20.1 cells.

Collectively, these results indicate that Tβ4 promotes MBP expressionthrough upregulation of miR-146a.

We also discovered unexpectedly that Tβ4 treatment and miR-146atransfection induces differentiation of OPC to mature oligodendrocytes.Rat primary embryonic OPCs and mouse N20.1 cells (n=3) were transfectedwith control (mock) and miR-146a vector, and treated with and withoutTβ4 (100 ng/ml). The OPCs were immunofluorescently stained withantibodies against MBP and counter-stained with DAPI. These data werequantified by counting the number of MBP positive cells. DAPI positivecells were considered as the total number of cells. FIG. 17 showsimmunohistochemistry of MBP in primary rat embryonic OPCs mouse N20.1cells. The primary rat embryonic OPCs (left panel) and N20.1 cells(right panel) were transfected with control vector (control), controlcells treated with Tβ4 (100 ng/ml) (Tβ4 (100 ng/ml)). Similarly, OPCswere also transfected with miR-146a (miR-146a) and miR-146a transfectedcells treated with Tβ4 (100 ng/ml) (Tβ4+146a). The cells wereimmunofluorescence stained with Cy3 labeled antibody against OLmarker—MBP and counterstained with DAPI. Images are merged (Merged).FIG. 18 shows the results of quantitative analysis of MBP positive cellsin primary rat embryonic OPCs and mouse N20.1 cells. Primary ratembryonic OPCs and mouse N20.1 cells were transfected with controlvector (control) and miR-146a vector (miR-146a transfection) followed bytreated without and with Tβ4 (Tβ4 (100 ng/ml) and miR-146atransfection+Tβ4 (100 ng/ml)). MBP positive cells afterimmunofluorescence staining were quantified by cell counting. Bar graphindicates percentage of MBP positive cells in primary rat embryonic OPCsand mouse N20.1 cells when DAPI positive cells was considered as 100%i.e. total number of cells. P<0.05 was considered as significant. Thenumber of MBP positive OPCs was significantly increased after treatmentwith Tβ4 or transfection with miR-146a in rat primary embryonic OPCs andmouse N20.1 cells (FIGS. 17 and 18, respectively. The miR-146atransfection amplified the effect of Tβ4 treatment on MBP immunostainingof both sets of OPCs. These data suggest that Tβ4 treatment and miR-146atransfection induced OL differentiation in both rat primary embryonicOPCs and mouse N20.1 cells.

In our work, we discovered unexpectedly that Tβ4 regulates miR-146a andfurther indicates that Tβ4 mediates oligodendrogenesis. Our datademonstrate that Tβ4 increases expression of miR-146a in rat primaryOPCs and mouse N20.1 OPCs, attenuates expression of IRAK and TRAF6, andreduces expression of phosphorylation/activation of ERK1, JNK1 andc-Jun, a negative regulator of MBP. Therefore, our data indicate thatTβ4 mediated oligodendrogenesis results from miR-146a suppression of theTLR proinflammatory pathway and modulation of the p38 MAPK pathway.

The innate immune system is an evolutionary primitive immediateresponsive defensive strategy that responds to pathogens by patternrecognition, and in contrast to the adaptive immune system, does notconfer long lasting immunity. The innate immune system orchestratesshort term responses to tissue injury resulting from proteins releasedby damaged or dying cells. TLRs are activated by numerous ligands frombacterial or viral pathogens, however, oxidized proteins, matrixproteins and cell adhesion proteins also activate the innate systemcontributing to disease processes such as atherosclerosis and acutecoronary syndromes. Inflammation initiates tissue repair after injury,however, it must be highly regulated so as not to harm the healing orrecovering tissue. Negative regulation of the innate immune system isachieved by several proteins and miRs. MiR-146a, is an importantnegative regulator of the innate immune system, and it is also found tobe highly expressed in developing oligodendrocytes duringdifferentiation. Therefore, our finding that Tβ4 upregulates miR-146a inour in vitro models of OPCs in conjunction with previous observationsthat Tβ4 promotes recovery after neurological injury suggest amultipurpose role of Tβ4 in promoting oligodendrocyte differentiation aswell as modulating the inflammatory response of the innate immune systemby downregulating two components of the pathway, IRAK1 and TRAF6.

Without limitation to any particular mechanism of action, theobservation that miR-146 is highly expressed in oligodendrocyte lineagecells indicates that maturation of oligodendrocytes occurs in anenvironment in which chronic inflammation is downregulated. Our resultsshowing that Tβ4 increases expression of miR-146a while promotingdifferentiation of OPCs to MBP positive oligodendrocytes, support this.Inhibiting miR-146a in Tβ4 treated cells removed the inhibitor effect onthe expression of IRAK and TRAF6 with no increase in MBP, expressionsuggesting that the miR-146a is a necessary component for MBP expressionand downregulation of the TLR proinflammatory pathway. Moreover,overexpression of miR-146a in Tβ4 treated cells showed an amplified MBPexpression and well as suppression of IRAK and TRAF6.

Our finding of Tβ4 modulation of the two key proinflammatory cytokines,IRAK and TRAF6 with corresponding down regulation of the expression ofphosphorylation/activation of ERK1, JNK1 and c-Jun indicates that Tβ4reduces inflammation, modulates the MAPKs and creates an environment foroligodendrocyte differentiation. Our work demonstrates similar resultsin rat primary OPCs, indicated that Tβ4 regulation of the MAPKs promotesoligodendrocyte differentiation. Furthermore, our data indicate thatupregulation of miR-146a influences activation of p38 MAPK andcorresponding suppression of ERK1 and JNK1, and thus promotesdifferentiation of OPCs to mature MBP oligodendrocytes.

In summary, we have discovered unexpectedly that Tβ4 treatmentupregulates miR-146a expression in rat primary embryonic OPCs and mouseN20.1 cells. Tβ4 treatment induced miR-146a suppression of theproinflammatory cytokines IRAK1 and TRAF6 leading to upregulation ofp38MAPK and inhibition of p-c-Jun, a negative regulator of MBP promoter.Tβ4 regulates miR-146a and may be required for MBP expression. Theseresults provide further support for the therapeutic use of Tβ4 tomediate oligodendrogenesis as a treatment for neurological injury,damage, or disease.

Thus, without limitation and without waiver or disclaimer of embodimentsor subject matter, some embodiments provide methods, systems, andcompositions which provide, increase, or promote miRNA-146a and provide,increase, or improve neuronal differentiation, oligodendrocytedifferentiation, and/or neurological outcome or function in treatedsubjects. Some embodiments comprise administration of a compositioncomprising a pharmaceutically effective amount of one or more ofmicroRNA-146a, a promoter of microRNA-146a expression, exosomescontaining or enriched in microRNA 146a, a microRNA-146a mimic, thymosinbeta 4, and a phosphodiesterase 5 inhibitor (the above referred toconvenience only as “miR-146a-related composition(s)” or“miR-146a-related composition administration”) to prevent, control, oralleviate neurological conditions, disease, or injuries in subjectsneeding such treatment. In accordance with some embodiments, withoutlimitation, one may inhibit such illness or injury throughmiR-146a-related composition administration for a finite interval oftime, thereby limiting the development or course of such condition,disease or injury.

In accordance with some embodiments, there is a high likelihood that theduration of therapy comprising miR-146a-related compositionadministration would be relatively brief and with a high probability ofsuccess. Prophylactic miR-146a-related composition administration ofsome embodiments may greatly reduce the incidence of damage associatedwith some forms of illness or injury.

Any appropriate routes of miR-146a-related composition administrationknown to those of ordinary skill in the art may comprise someembodiments.

MiR-146a-related compositions of some embodiments would be administeredand dosed in accordance with good medical practice, taking into accountthe clinical condition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. In accordancewith some embodiments, experience with dose levels in animals is knownand dose levels acceptable for safe human use are determinable orscalable in accordance with such information and/or good medicalpractice. The “pharmaceutically effective amount” for purposes herein isthus determined by such considerations as are known in the art. Theamount must be effective to achieve improvement, including but notlimited to, decrease in damage or injury, or improvement or eliminationof symptoms and other indicators as are selected as appropriate measuresby those skilled in the art.

In accordance with some embodiments, miR-146a-related compositions canbe administered in various ways. They can be administered alone or as anactive ingredient in combination with pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles. The miR-146a-relatedcompositions can be administered orally, subcutaneously or parenterallyincluding intravenous, intraarterial, intramuscular, intraperitoneal,and intranasal administration as well as intrathecal and infusiontechniques, or by local administration or direct inoculation to the siteof disease or pathological condition. Implants of the miR-146a-relatedcompositions may also be useful. The patient being treated is awarm-blooded animal and, in particular, mammals including humans. Thepharmaceutically acceptable carriers, diluents, adjuvants and vehiclesas well as implant carriers generally refer to inert, non-toxic solid orliquid fillers, diluents or encapsulating material not reacting with theactive components of some embodiments. In some embodiments,miR-146a-related compositions may be altered by use of antibodies tocell surface proteins or ligands of known receptors to specificallytarget tissues of interest.

Since the use of miR-146a-related composition administration inaccordance with some embodiments specifically targets the evolution,expression, or course of associated conditions or pathologies, it isexpected that the timing and duration of treatment in humans mayapproximate those established for animal models in some cases.Similarly, the doses established for achieving desired effects usingsuch compounds in animal models, or for other clinical applications,might be expected to be applicable in this context as well. It is notedthat humans are treated generally longer than the experimental animalsexemplified herein which treatment has a length proportional to thelength of the disease process and drug effectiveness. The doses may besingle doses or multiple doses over periods of time. The treatmentgenerally has a length proportional to the length of the disease processand drug effectiveness and the patient species being treated.

In some embodiments, subjects, for example a human subject, having aneurological disease, disorder, condition or injury, may be treated witha composition comprising exosomes containing miR-146a. In someembodiments, the subject is administered exosomes containing miR-146aderived from naïve cells that are capable of producing miR-146acontaining exosomes, for example, stem cells, (for example, apluripotent stem cell, for example, an embryonic stem cell; an inducedpluripotent stem cell; a hair follicle stem cell; a hematopoietic stemcell; a very small embryonic like stem cell, a mesenchymal stem cell; anendometrial regenerative cell (ERC); or a progenitor cell), mesenchymalstromal cells, umbilical cord cells, endothelial cells, Schwann cells,hematopoietic cells, reticulocytes, monocyte-derived dendritic cells(MDDCs), monocytes, B lymphocytes, antigen-presenting cells, glialcells, astrocytes, neurons, oligodendrocytes, spindle neurons,microglia, or mastocytes. In some embodiments, cells recited above thatare capable of producing miR-146a containing exosomes are furthergenetically manipulated to express additional or higher amounts ofmiR-146a, (by transfecting cells transiently or stably with vectors orpolynucleotides encoding miR-146a or complementary sequences thereof) asthese exosomes are said to be “enriched” in miR-146a microRNA.

When administering the miR-146a-related compositions parenterally, insome embodiments, the composition may be formulated in a unit dosageinjectable form (solution, suspension, or emulsion). The pharmaceuticalformulations suitable for injection (for example, I.V. or I.P.) includesterile aqueous solutions or dispersions and sterile powders forreconstitution into sterile injectable solutions or dispersions. Thecarrier can be a solvent or dispersing medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), suitable mixtures thereof, andvegetable oils.

When necessary, proper fluidity can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredsize in the case of dispersion and by the use of surfactants.Non-aqueous vehicles such a cottonseed oil, sesame oil, olive oil,soybean oil, corn oil, sunflower oil, or peanut oil and esters, such asisopropyl myristate, may also be used as solvent systems for suchmiR-146a-related composition compositions. Additionally, variousadditives which enhance the stability, sterility, and isotonicity of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. In many cases, it will be desirable to include isotonicagents, for example, sugars, sodium chloride, and the like. Prolongedabsorption of the injectable pharmaceutical form can be brought about bythe use of agents delaying absorption, for example, aluminummonostearate and gelatin. According to some embodiments, however, anyvehicle, diluent, or additive used would have to be compatible with themiR-146a-related compositions.

Sterile injectable solutions can be prepared by incorporatingmiR-146a-related compositions utilized in practicing the someembodiments in the required amount of the appropriate solvent withvarious of the other ingredients, as desired.

A pharmacological formulation of some embodiments may be administered tothe patient in an injectable formulation containing any compatiblecarrier, such as various vehicle, adjuvants, additives, and diluents; orthe inhibitor(s) utilized in some embodiments may be administeredparenterally to the patient in the form of slow-release subcutaneousimplants or targeted delivery systems such as monoclonal antibodies,vectored delivery, iontophoretic, polymer matrices, liposomes, andmicrospheres. Many other such implants, delivery systems, and modulesare well known to those skilled in the art.

In some embodiments, without limitation, the miR-146a-relatedcompositions may be administered initially by intravenous injection tobring blood levels of miR-146a to a suitable level. The patient's levelsof miR-146a are then maintained by injection, infusion or IV dosageforms comprising exosomes of the present invention, although other formsof administration, dependent upon the patient's condition and asindicated above, can be used. The quantity to be administered and thetiming of administration may vary for the patient being treated. In someembodiments, the frequency of dosing can be determined by a physician orclinician using standard dosing methods known in the art. For example,the subject may be given an initial dosage that differs from latermaintenance dosage forms. In some embodiments, the subject may beadministered a first dose, and then the second and subsequent doses maybe adjusted up or down in accordance with various factors, for example,the subject's tolerance to the administered doses, the overall effectprovided by the last dose, the presence or absence of adverse sideeffects, the severity of the neurological disease, condition, disorderor injury and other factors known in the art. Generally speaking, one ormore doses may be administered per day, or per week, or per month, withintervals ranging from 1 day to about 7 days, to about 14 days, or toabout 30 days.

In various embodiments, exemplary compositions of the invention can bedelivered to the subject at a dose that is effective to treat and/orprevent a neurological disease, disorder, condition or injury. Theeffective dosage will depend on many factors including the gender, age,weight, and general physical condition of the subject, the severity ofthe pain, the particular compound or composition being administered, theduration of the treatment, the nature of any concurrent treatment, thecarrier used, and like factors within the knowledge and expertise ofthose skilled in the art. As appropriate, a treatment effective amountin any individual case can be determined by one of ordinary skill in theart by reference to the pertinent texts and literature and/or by usingroutine experimentation (see, e.g., Remington, The Science and Practiceof Pharmacy (21^(st) ed. 2005)). In one embodiment, the amount ofmiR-146a present in the exosomes administered to the subject in needthereof can range from about 0.1 to about 10.0 mg/m² surface area of thepatient, e.g., about 0.6 to about 4.0 mg/m², about 1.0 to about 3.0mg/m², or about 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6,2.8, 3.0, 3.2, 3.4, 3.6, 3.8, or 4.0 mg/m². In some instances, the dosecan be even lower, e.g., as low as 0.1, 0.05, 0.01, 0.005, or 0.001mg/m² or lower. In some instances, the dose can be even higher, e.g., ashigh as 20, 50, 100, 500, or 1000 mg/m² or higher. The present inventionencompasses every sub-range within the cited ranges and amounts.

In some embodiments, the compositions comprising exosomes containingmiR-146a of the invention may be administered at doses ranging fromabout 1×10¹ to about 1×10¹⁵ exosomes per kg body weight of the patient,or about 1×10³ to about 1×10¹⁴ exosomes per kg body weight of thepatient, or about 1×10³ to about 1×10¹³ exosomes per kg body weight ofthe patient, or about 1×10³ to about 1×10¹² exosomes per kg body weightof the patient, or about 1×10³ to about 1×10¹¹ exosomes per kg bodyweight of the patient, or about 1×10³ to about 1×10¹⁰ exosomes per kgbody weight of the patient, or about 1×10³ to about 1×10⁹ exosomes perkg body weight of the patient, or about 1×10³ to about 1×10⁸ exosomesper kg body weight of the patient, or about 1×10³ to about 1×10⁷exosomes per kg body weight of the patient, or from about 1×10³ to about1×10⁶ exosomes per kg body weight of the patient, or from about 1×10⁷ toabout 1×10¹⁵ exosomes per kg body weight of the patient, or from about1×10⁷ to about 1×10¹⁴ exosomes per kg body weight of the patient, orfrom about 1×10⁷ to about 1×10¹³ exosomes per kg body weight of thepatient, or from about 1×10⁷ to about 1×10¹² exosomes per kg body weightof the patient.

In some embodiments, a subject in need of treatment can be administeredwith a dose of about 1×10⁷ to about 1×10¹⁵ exosomes per dose, or perday, or as a bolus. Preferably, the exosomes (containing or enriched inmiR-146a) containing composition or formulation of the invention isadministered at a dose of about 1×10⁹ to about 1×10¹⁵ exosomes per kgbody weight of the patient, or a dose (a therapeutically effective dose,or a daily dose, or a weekly dose) of about 1×10¹⁰ to about 5×10¹³exosomes for an average adult weighing approximately 70 kg.

In various embodiments, the exemplified doses of exosomes per kg weightof the patient are daily doses or therapeutically effective doses,either in unit form or in sub-unit forms to be dosed one or more timesper day. In various embodiments, each of these daily doses ortherapeutically effective doses, either in unit form or in sub-unitforms may be administered to a subject with a neurological disease,disorder, condition or injury in the form of parenteral administration,for example, intravenous (I.V.), intraperitoneal (I.P.), infusion, orstereotactically administered in the form of a liquid, for example, asolution, an emulsion, or a suspension containing the exosomes and oneor more pharmaceutically acceptable excipients, carriers, or diluents.

In some embodiments, a subject with a neurological disease, injury,disorder or condition may be treated with a pharmaceutically effectiveamount of the exosomes of the present invention, for example, thesubject may be administered with 1×10³ to about 1×10¹⁵ exosomes perdose, or per day, or as a bolus. In some embodiments, compositions ofthe present invention containing from about 1×10³ to about 1×10¹⁵exosomes, can be administered parenterally to a subject in need thereof,once every two days, for about 1-4 weeks and then once per week, for thenext 4 to about 52 weeks, or until a skilled physician or cliniciandetermines that treatment can be discontinued, or until symptoms haveresolved.

In some embodiments, patients with a neurological disease, injury ordisorder, for example, a stroke or traumatic brain injury, or peripheralneuropathy and diabetic neuropathy patient, may be treated byadministration of an initial or first dose of about 4×10¹¹-about 5×10¹³exosomes/kg body weight of the subject, delivered at 24 hours afterinjury. Follow-on doses may range from about 5×10⁹ to about 5×10¹³exosomes/kg body weight of the subject, 14 days after the initial dose,optionally with a single weekly dose of about 5×10⁹ to about 5×10¹³exosomes per week thereafter until dosing is terminated. In someembodiments, multiple sclerosis patients may be treated by dosing 4×10¹¹to about 5×10¹³ exosomes/kg body weight of the subject, twice weekly forabout 1-8 weeks, preferably 1-4 weeks in duration.

Additionally, in some embodiments, without limitation, miR-146a-relatedcompositions may be administered in situ to bring internal levels to asuitable level. The patient's levels are then maintained as appropriatein accordance with good medical practice by appropriate forms ofadministration, dependent upon the patient's condition. The quantity tobe administered and timing of administration may vary for the patientbeing treated.

SEQUENCES  SEQ ID NO: 1:  MicroRNA-146a, described generally as: UGAGAACUGAAUUCCAUGGGUU  SEQ ID NO: 2: Thymosin beta 4, described generally as: Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu-Ile-Glu-Lys-Phe-Asp-Lys-Ser-Lys-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Glu-Lys-Asn-Pro-Leu-Pro-Ser-Lys-Glu-Thr-Ile-Glu-Gln- Glu-Lys-Gln-Ala-Gly-Glu-Ser  SEQ ID NO: 3: 5T-TACTICGGCTGGITCCTGAC-3T 5T-  SEQ ID NO: 4: 5T-GCCTICCCGTAGICACAAAA-3T 5T  SEQ ID NO: 5: ATGGCATCACAGAAGAGACCCTCA-3' 5T-  SEQ ID NO: 6: TAAAGAAGCGCCCGATGGAGTCAA-3' 5T-  SEQ ID NO: 7:  ATGACGAAATGACCGGCTAC-3'5T-  SEQ ID NO: 8:  ACAACGTTCTTCCGGTCAAC-3 5T-  SEQ ID NO: 9: GCTGAACAAAGGGAGAGACG-3T 5T-  SEQ ID NO: 10: TGCTTTCTCCCCAAATTGAC-3T 5T-  SEQ ID NO: 11: TICAATGICCACAGATCCGA-3T 5'-  SEQ ID NO: 12: CTAACCAATTCCCCATCCCT-3T 5T-  SEQ ID NO: 13: GCCATTCTGGTAGAGGAAGTTTCTC-3' 5T-  SEQ ID NO: 14: CGCCAGTCCAAAATCAAGAATC-3' 5T-  SEQ ID NO: 15:  TGAAGCAGAGCATGACCTTG-3'5T-  SEQ ID NO: 16:  CACAAGAACTGAGTGGGGGT-3' 5T-  SEQ ID NO: 17: CGCAACCAGTCAAGTTCTCA-3T  SEQ ID NO: 18:  GAAAAGTAGCCCCCAACCTC-3T 

Example 3

Cerebral Endothelial Cell Exosomes Enriched in miR-146a for theTreatment of Neurological Diseases, Injuries, and Disorders

To investigate the effect of miR-146a exosomes on diabetic neuropathy,male diabetic mice (db/db) at age of 20 weeks were treated once a weekwith miR-146a containing exosomes derived from mesenchymal stromal cells(MSCs) miR-146a exosomes (10¹⁰ exosomes/mouse, n=8/group) or saline for4 consecutive weeks via a tail vein. Non-diabetic mice (dm) wereemployed as a negative control group. Behavioral tests andelectrophysiological tests (sensory and motor nerve conduction velocity)were performed once a week or every two weeks, respectively. The animalswere sacrificed 8 weeks after the initial administration.

The inventors of the present disclosure examined whether administrationof tailored miR-146a exosomes affects miR-146a levels in diabeticanimals using a dosing regimen shown in FIG. 19. Sciatic nerve tissuesand blood sera were collected 24 hours after the last treatment.Quantitative real-time RT-PCR data show levels of miR-146a weredecreased in sera (FIG. 20A) and sciatic nerves (FIG. 20B) of diabeticmice compared with tissues of non-diabetic (dm) mice. However, db/db(db) mice treated with tailored miR-146a-exosomes displayed significantelevation of miR-146a levels (db+exo miR-146a). N=3/group. #P<0.05 vscontrol dm mice, **p<0.01 vs diabetic mice treated with saline.

Next, the inventors examined the effect of miR-146a-exosomes onneurological function including electrophysiological tests, thermalhyperalgesia and tactile allodynia measurements in diabetic mice.Sciatic nerve conduction velocity (FIG. 21A) was examined usingorthodromic recording techniques. To examine the sensitivity of mice toheat, plantar test was done using a thermal stimulation meter. As shownabove in FIGS. 21B and 21C, we found that exosomes had a statisticallysignificant positive impact on each of these measures at various timesafter administration.

We next measured the impact of miR-146a exosomes on myelin thickness inthe sciatic nerve. Representative images of semi-thin toluidineblue-stained transverse sections of sciatic nerves derived fromnon-diabetic (dm, FIG. 22A), diabetic mice treated with saline (db, FIG.22B) or diabetic mice treated with tailored miR-146a-exosomes (db+exomiR-146a, FIG. 22C). The quantitative data of histomorphometricparameters of sciatic nerves are shown in tabular form in FIG. 22D.These results showed that mean fiber and axon diameters as well asmyelin sheath thickness were reduced in diabetic mice, neverthelessincreased in tailored miR-146a-exosomes treated diabetic mice. Meang-ratio was significantly increased in diabetic animals indicating mildhypomyelination whereas tailored miR-146a exosome treatment led to asignificant increase of myelinated fiber density. Bar=50 μm. N=8/group.

Compared to age matched non-diabetic db/m mice, db/db mice treated withsaline showed approximately a 25% decrease in the number ofintraepidermal nerve fibers (IENF) in skin of the foot-pads identifiedby the antibody against PGP9.5, which is a cytoplasmic ubiquitinC-terminal hydrolase expressed in all types of efferent and afferentperipheral nerve fibers.(See FIG. 23). In contrast, the treatment withtailored miR-146a-exosomes significantly increased PGP9.5 immunoreactiveintraepidermal nerve fiber (arrows) in diabetic mice (db+exo miR-146a)by representative microscopic images and quantitative analysis as shownin FIG. 24. Bar=50 μm. ## P<0.01 versus the dm group, * P<0.05 versus dbgroup. n=8/group.

Dicer is essential for mature miRNA formation. Real-time RT-PCR analysisshowed that ablation of Dicer in neural stem cells of adult mice (AdultAscl1creER™/Dicer^(fl/fl)/Tomato^(fl/fl) (Dicer−/−) led to significantreduction of miR-146a and among others compared to wild-type mice(Ascl1creER™/Tomato^(fl/fl) (Dicer+/+) See FIG. 25). However, as shownin FIG. 25, intranasal administration of cerebral endothelial cellderived exosomes (1×10⁹ particles/administration) every other day forfive times significantly increased miR-146a levels in neural stem cellsof Dicer knockout mice (n=10) compared to Dicer knockout mice treatedwith saline (n=12).

While some embodiments have been particularly shown and described withreference to the foregoing preferred and alternative embodiments, itshould be understood by those skilled in the art that variousalternatives to the embodiments described herein may be employed inpracticing the invention without departing from the spirit and scope ofthe invention as defined in the following claims. It is intended thatthe following claims define the scope of the invention and that themethods, systems, and compositions within the scope of these claims andtheir equivalents be covered thereby. This description of someembodiments should be understood to include all novel and non-obviouscombinations of elements described herein, and claims may be presentedin this or a later application to any novel and non-obvious combinationof these elements. The foregoing embodiments are illustrative, and nosingle feature or element is essential to all possible combinations thatmay be claimed in this or a later application. Where the claims recite“a” or “a first” element of the equivalent thereof, such claims shouldbe understood to include incorporation of one or more such elements,neither requiring nor excluding two or more such elements.

What is claimed is:
 1. A method of treating a subject suffering from aneurological disease or injury with exosomes containing miR-146amicroRNA, comprising: administering to the subject in need thereof, atherapeutically effective amount of the exosomes containing miR-146amicroRNA to treat the subject with the neurological disease or injury.2. The method of claim 1, wherein the exosomes are derived from one ormore exosome producing cell populations comprising: stem cells,mesenchymal stromal cells, umbilical cord cells, endothelial cells,Schwann cells, hematopoietic cells, reticulocytes, monocyte-deriveddendritic cells (MDDCs), monocytes, B lymphocytes, antigen-presentingcells, glial cells, astrocytes, neurons, oligodendrocytes, spindleneurons, microglia, or mastocytes.
 3. The method of claim 2, wherein thestem cells are selected from the group consisting of: progenitor cells;pluripotent stem cells; embryonic stem cells; induced pluripotent stemcells; hair follicle stem cells; hematopoietic stem cells; very smallembryonic like stem cells; mesenchymal stem cells; endometrialregenerative cells (ERC); and neural progenitor cells.
 4. The method ofclaim 1, wherein the exosomes are administered to the subjectintravenously, nasally, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intratracheally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, injection, infusion, continuous infusion,localized perfusion bathing target cells directly, via a catheter, via alavage, or stereotactically into neural tissue.
 5. The method of claim1, wherein the cell population is derived from the subject.
 6. Themethod of claim 1, wherein the neurological disease or injury comprisesstroke, brain injury, central pontine, dementia, multiple sclerosis (MS)(together with the similar diseases called idiopathic inflammatorydemyelinating diseases), tumefactive multiple sclerosis, Solitarysclerosis, cognitive decline from aging, Alzheimer's disease,Parkinson's disease, epilepsy, migraine, neuropathy, for example,peripheral neuropathy, Vitamin B12 deficiency, myelinolysis, TabesDorsalis, transverse myelitis, Devic's neuromyelitis optica, fulminantor acute idiopathic inflammatory-demyelinating disease, Marburg variantof multiple sclerosis, Baló's concentric sclerosis, Schilder's disease,acute disseminated encephalomyelitis; transverse myelitis, opticneuritis, progressive multifocal leukoencephalopathy, acute hemorrhagicleukoencephalitis, acute disseminated encephalomyelitis, anti-myelinoligodendrocyte glycoprotein autoimmune encephalomyelitis,Leukodystrophy, adrenoleukodystrophy, adrenomyeloneuropathy, chronicinflammatory demyelinating polyradiculoneuropathy (CIDP), Guillain-Barresyndrome, chronic inflammatory demyelinating polyneuropathy, or anti-MAGperipheral neuropathy.
 7. The method of claim 6, wherein theneurological disease or injury comprises dementia, traumatic braininjury, peripheral neuropathy, diabetic neuropathy, stroke or multiplesclerosis.
 8. The method of claim 1, wherein the subject is administeredwith about 1×10⁷ to about 1×10¹⁵ exosomes per dose, or per day, or as abolus.
 9. A method of treating a subject suffering from a neurologicaldisease or injury with cells capable of producing miR-146a microRNA, themethod comprising: providing a cell population capable of producingexosomes containing miR-146a; and administering to the subject in needthereof, the cell population capable of producing exosomes containingmiR-146a microRNA in a pharmaceutically effective amount to treat thesubject with respect to the neurological disease or injury.
 10. Themethod of claim 9, wherein the cell population comprises: stem cells,mesenchymal stromal cells, umbilical cord cells, endothelial cells,Schwann cells, hematopoietic cells, reticulocytes, monocyte-deriveddendritic cells (MDDCs), monocytes, B lymphocytes, antigen-presentingcells, glial cells, astrocytes, neurons, oligodendrocytes, spindleneurons, microglia; or mastocytes.
 11. The method of claim 10, whereinthe stem cells are selected from the group consisting of: embryonic stemcells; pluripotent stem cells; induced pluripotent stem cells; hairfollicle stem cells; hematopoietic stem cells; very small embryonic likestem cells; mesenchymal stem cells; endometrial regenerative cells(ERC); and progenitor cells.
 12. The method of claim 9, wherein the cellpopulation is administered stereotactically in the brain.
 13. The methodof claim 9, wherein the neurological disease or injury comprises stroke,brain injury, central pontine, dementia, multiple sclerosis (MS)(together with the similar diseases called idiopathic inflammatorydemyelinating diseases), tumefactive multiple sclerosis, Solitarysclerosis, cognitive decline from aging, Alzheimer's disease,Parkinson's disease, epilepsy, migraine, neuropathy, for example,peripheral neuropathy, Vitamin B12 deficiency, myelinolysis, TabesDorsalis, transverse myelitis, Devic's neuromyelitis optica, fulminantor acute idiopathic inflammatory-demyelinating disease, Marburg variantof multiple sclerosis, Baló's concentric sclerosis, Schilder's disease,acute disseminated encephalomyelitis; transverse myelitis, opticneuritis, progressive multifocal leukoencephalopathy, acute hemorrhagicleukoencephalitis, acute disseminated encephalomyelitis, anti-myelinoligodendrocyte glycoprotein autoimmune encephalomyelitis,Leukodystrophy, adrenoleukodystrophy, adrenomyeloneuropathy, chronicinflammatory demyelinating polyradiculoneuropathy (CIDP), Guillain-Barresyndrome, chronic inflammatory demyelinating polyneuropathy, or anti-MAGperipheral neuropathy.
 14. The method of claim 13, wherein theneurological disease or injury comprises dementia, brain injury,peripheral neuropathy, stroke or multiple sclerosis.
 15. A method oftreating a subject suffering from dementia, brain injury, neuropathy,stroke or multiple sclerosis with exosomes containing miR-146a microRNA,the method comprising: administering to the subject in need thereof, theexosomes containing miR-146a microRNA in a pharmaceutically effectiveamount to treat the subject with the dementia, brain injury, neuropathy,stroke or multiple sclerosis.
 16. The method according to claim 15,wherein the exosomes are derived from cell populations geneticallymodified to produce miR-146a in amounts greater than naïve un-modifiedcells.
 17. The method of claim 15, wherein the exosomes are derived fromone or more cell populations comprising: stem cells, mesenchymal stromalcells, umbilical cord cells, endothelial cells, Schwann cells,hematopoietic cells, reticulocytes, monocyte-derived dendritic cells(MDDCs), monocytes, B lymphocytes, antigen-presenting cells, glialcells, astrocytes, neurons, oligodendrocytes, spindle neurons,microglia, or mastocytes.
 18. The method of claim 17, wherein the stemcells are selected from the group consisting of: embryonic stem cells;pluripotent stem cells; induced pluripotent stem cells; hair folliclestem cells; hematopoietic stem cells; very small embryonic like stemcells; mesenchymal stem cells; endometrial regenerative cells (ERC); andprogenitor cells.
 19. The method of claim 15, wherein the exosomes areadministered intravenously, nasally, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intratracheally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, injection, infusion, continuous infusion,localized perfusion bathing target cells directly, via a catheter, via alavage, or directly into neural tissue.
 20. A method of treating asubject suffering from stroke with exosomes containing miR-146a themethod comprising: administering to the subject in need thereof, theexosomes in a pharmaceutically effective amount to treat the subjectwith the stroke.
 21. The method of claim 20, wherein the exosomes arederived from a cell population comprising: stem cells, mesenchymalstromal cells, umbilical cord cells, endothelial cells, Schwann cells,hematopoietic cells, reticulocytes, monocyte-derived dendritic cells(MDDCs), monocytes, B lymphocytes, antigen-presenting cells, glialcells, astrocytes, neurons, oligodendrocytes, spindle neurons,microglia, or mastocytes.
 22. The method of claim 21, wherein the stemcells are selected from the group consisting of: embryonic stem cells;pluripotent stem cells; induced pluripotent stem cells; hair folliclestem cells; hematopoietic stem cells; very small embryonic like stemcells; mesenchymal stem cells; endometrial regenerative cells (ERC); andprogenitor cells.
 23. The method of claim 20, wherein the exosomes areadministered intravenously, nasally, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intratracheally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, injection, infusion, continuous infusion,localized perfusion bathing target cells directly, via a catheter, via alavage, or directly into neural tissue.